universidade federal do rio de janeiro instituto de química programa

Transcrição

UNIVERSIDADE FEDERAL DO RIO DE JANEIRO
INSTITUTO DE QUÍMICA
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIA DE ALIMENTOS
LAIDSON PAES GOMES
ESTUDO DO EFEITO DA SONICAÇÃO NA PRODUÇÃO DE BIOPLÁTICOS DE
QUITOSANA: ATIVIDADE ANTIMICROBIANA E COMPORTAMENTO
REOLÓGICO
Rio de janeiro
2015
Laidson Paes Gomes
REOLÓGICO
Tese de Doutorado apresentada ao Programa
de Pós-Graduação em Ciência de Alimentos da
Universidade Federal do Rio de Janeiro, como
requisito parcial à obtenção do título de
Doutorado em Ciências.
Orientadores:
Prof.a Dra Vânia M. Flosi Paschoalin
Prof.° Dr° Eduardo M. Del Aguila
Rio de Janeiro
2015
LAIDSON PAES GOMES
REOLÓGICO
Tese de doutorado apresentada ao programa de pós-graduação em Ciência de Alimentos da
Universidade Federal do Rio de Janeiro, como parte dos requisitos para obtenção do grau de
Doutor em Ciências.
Aprovada em:26 de novembro de 2015.
Banca Examinadora
Prof.a Dra Vânia M. Flosi Paschoalin-IQ/UFRJ
(Orientadora)
Prof.° Dr° Eduardo M. Del Aguila-IQ/UFRJ
(Coorientador)
Prof.a Dra Maria H. Rocha Leão- EQ/UFRJ
Prof.a Dra Lourdes M. Correa Cabral-EMBRAPA
Prof.a Dra Rossana M. da Silva Moreira Thiré-COPPE/UFRJ
Prof.° Dr° Lucio Mendes Cabral-FF/UFRJ
AGRADECIMENTOS
A minha família, Nellio R. B. Gomes (pai), Jussiara P. Gomes (mãe) e Thaiga P. Gomes
(irmã) pelo imenso apoio em todos esses anos de estudo e principalmente por ser a referência
de educação, caráter, honestidade e profissionalismo fundamental para minha formação
pessoal, permitindo chegar a esse estágio.
Ao prof° Joab T Silva (in memorian) pela sua incapacidade de dizer não aos alunos em busca
de estágio, que após de diversas tentativas em diferentes laboratórios foi o primeiro a me dizer
“sim” e permitir que eu iniciasse minha carreira cientifica.
A minha orientadora profa Vânia M. F. Paschoalin pelos esforços depositados em mim, dando
todo auxílio possível para o meu desenvolvimento e crescimento profissional, principalmente
pela confiança no meu trabalho e no meu potencial, permitindo a minha participação na
elaboração de projetos de pesquisa e do desenvolvimento dos mesmos em diferentes grupos
de pesquisas sediados em variados países (Brasil, Portugal e Inglaterra).
A meu orientador e amigo prof° Eduardo M. Del Aguila, por esses oito anos de orientação e
amizade, onde com toda paciência me ensinou na prática o que é e como se faz ciência. Além
de me apoiar e ajudar a enfrentar todas as etapas difíceis no decorrer desses anos de
convivência.
A profa Cristina T. Andrade por todos esses anos de parceria bem produtivos, pela confiança e
indicação para trabalhar com grupo de pesquisa na Universidade do Porto e pela
disponibilidade de todos os seus orientados em me ajudar.
A profa Maria P. Gonçalves, por ser essa pessoa fantástica, dona de muito conhecimento e
generosidade, pela ótima recepção e auxilio tornando simples e rápida a adaptação a
Universidade do Porto, por todas as historias e curiosidade sobre Portugal antes da criação do
Brasil durante nossos almoços e de toda orientação no desenvolvimento do projeto.
A Hiléia K. S. Souza e José M. Campiña por me recebem como uma família, tornando minha
adaptação muito fácil a Cidade e Universidade do Porto, além do empenho e parceria direta
no desenvolvimento do projeto.
Aos meus amigos Artur, Claudio, Diego, Gabriel, Leandro, Pedro e Thiago que sempre me
apoiaram e acreditaram no meu potencial, apesar de nunca entenderem o que eu realmente
fazia há tantos anos na faculdade, acreditando ser uma coisa muito legal e que era algo muito
difícil.
A todos os meus amigos que estão e que passaram pelo LAABBM (Analy, Giseli, Patrícia,
Cyntia, Karine, Val, Wilson, Julia, Diego, Davi e Rafael Luiz), que ajudaram no meu
desenvolvimento e crescimento profissional dentro desses oito anos, por tornarem o ambiente
de trabalho agradável e divertido e principalmente por aturarem minhas piadas e meu
temperamento muitas vezes “reclamão”
A Nathalia Sales por ser um ouvido incansável de minhas historias, pelas alegrias e tristezas
partilhadas e por me estimular e fazer parte das minhas evoluções durante “o segundo
semestre”.
As amizades que construí na cidade do Porto (Julia, Marina, Raissa, Norton, Muriel, Fernanda
e Bonna), que tornaram a adaptação à vida nova muito simples e divertida, além de estamos
sempre juntos nos apoiando e nos auxiliando nos momentos de dificuldade.
A Nastassia Porto por todo o tempo de convivo, por ser uma referência para o meu
crescimento, me fez acreditar no meu potencial e me estimulando a ser sempre maior,
mostrando um mundo de possibilidades e que eu era capaz de alcançar objetivos que
acreditava estarem muito distantes da minha realidade.
A Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro
(FAPERJ, Rio de Janeiro, Brazil), ao Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq, Brasília, Brasil), a Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior (CAPES, Brasília, Brasil) pelo Programa Institucional de Bolsas de Doutorado
Sanduíche no Exterior, PDSE; Proc nº 18935/12-5.
RESUMO
GOMES, Laidson Paes. Produção de nanopartículas de quitosana: sua atividade
antimicrobiana e a complexação de fibras/nanopartículas na produção de filmes. Rio de
Janeiro, 2015. Tese de (Doutorado em Ciência de Alimentos) Programa de Pós-Graduação em
Ciência de Alimentos, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 2015.
A aplicação de materiais em nano escala e nanoestruturas é uma área emergente, uma vez que
estes materiais podem proporcionar soluções para desafios tecnológicos e ambientais, com o
objetivo de preservar o meio ambiente e os recursos naturais. A quitosana é um polissacarídeo
natural obtido a partir da N-desacetilação da quitina. A quitosana vem sendo largamente
utilizada nas indústrias de alimentos e de bioengenharia, com proposito de encapsular
ingredientes ativos em alimentos, na imobilização de enzimas, e como um veículo para
libertação controlada de fármacos. As propriedades biológicas e químicas mais importantes
são a sua biodegradabilidade, biocompatibilidade e bioatividade. Nanopartículas à base de
quitosana vem sendo preparadas por diferentes métodos e várias aplicações vêm sendo
desenvolvidas para estes produtos. Neste estudo, avaliou-se a degradação de fibras de
quitosana (NS) em solução utilizando sonda de ultrassom durante diferentes períodos de
tempo (S(ts)= 5, 15 and 30min) formando NPs (nanoprtículas), que foram caracterizadas por
espalhamento de luz dinâmico (DLS), as soluções foram misturadas em diferentes proporções
NS/S(ts) 3:7, 1:1 e 7:3. A partir de cada mistura NS/S(ts), foram preparados filmes que
tiveram suas propriedades mecânicas, de barreira, sensibilidade à humidade, a transparência, a
permeabilidade ao vapor de água (WVP) e a cor dos biofilmes avaliados. A morfologia dos
filmes foi investigada por microscopia de força atômica (AFM) e microscopia eletrônica de
varredura (MEV). Os resultados indicam que, através do controle das condições de sonicação
e proporção da mistura, é possível ajustar a viscoelasticidade e aspecto morfológico das
misturas em níveis intermédios em relação aos seus componentes individuais, com a mesma
distribuição de graus de polimerização e sem a incorporação de aditivos ou plastificantes. Os
resultados mostraram que foi possível modificada e controlada WVP, propriedades mecânicas
e de barreira ao ajustar a proporção de mistura dos filmes produzidos. As análises cor e
opacidade demonstraram uma boa transparência dos filmes com L coordenar ≥ 91. As
imagens obtidas por AFM e SEM mostrou a presença de partículas em torno de 10-300 nm, e
uma boa morfologia para os filmes produzidos a partir das mistura. As soluções coloidais
mostraram partículas com raio hidrodinâmico (Rh) no intervalo de 220-3000 nm, e os valores
de potencial zeta na gama de 24-25 mV, demonstrando uma estabilidade moderada. As
atividades antimicrobianas da quitosana com diferentes distribuições de massa molar foram
testadas contra patógenos alimentares. Foi avaliado o potencial zeta, a concentração mínima
inibitória (MIC), a concentração mínima bacteriana (MBC), atividade antibacteriana in vitro e
a integridade da membrana celular foi investigada contra Escherichia coli. Os estudos
demonstram que as partículas de menor tamanho apresentam melhor atividade antimicrobiana
com mesmos valores para MIC e o MBC (0,2 mg/mL) sobre a bactéria testada Escherichia
coli.
Palavras-chave: Quitosana, Ultrassonicação, Nanopartículas, Biofilme, Atividade antimicrobiana.
ABTRACT
GOMES, Laidson Paes. Produção de nanopartículas de quitosana: sua atividade
antimicrobiana e a complexação de fibras/nanopartículas na produção de filmes. Rio de
Janeiro, 2015. Tese de (Doutorado em Ciência de Alimentos) Programa de Pós-Graduação em
Ciência de Alimentos, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 2015.
The use of nanoscale materials and nanostructures is an emerging area, since these
materials may provide solutions to technological and environmental challenges in order to
preserve the environment and natural resources. Chitosan is a natural polysaccharide prepared
by the N-deacetylation of chitin. It has been widely used in food and bioengineering
industries, including the encapsulation of active food ingredients, in enzyme immobilization,
and as a carrier for controlled drug delivery. The most significant biological and chemical
properties are its biodegradability, biocompatibility and bioactivity. Chitosan-based
nanoparticles have been prepared by various methods. Falta alguma coisa. In this study, it was
evaluated the chitosan-fibril (NS) degradation using sonication for different time of periods
(S(ts)=5, 15 and 30min), forming NP (nanoparticles) that were evaluated by dynamic light
scattering (DLS). Mixed solutions of NS/S(ts) at different ratios 3:7, 1:1 and 7:3 were used
for preparing blend films by the knife coating method. The NS/S(ts) films had their
mechanical, barrier properties, moisture sensitivity, transparency, water vapor permeability
(WVP) and color were evaluated. The morphology of films was investigated by atomic force
microscopy (AFM) and scanning electron microscopy (SEM). The results indicate that,
through the control of the sonication conditions and mixture ratio, it is possible to adjust the
viscoelasticity and morphological aspects of the mixtures at intermediate levels relative to
their individual components, with the same distribution of degrees of polymerization and
without the incorporation of additives or plasticizers. The results showed that it was possible
modified and controlled the WVP, mechanical and barrier properties by adjusting the mixing
ratio in the films produced. The color and opacity analyses demonstrated a good transparency
of films with L coordinate ≥ 91. The images obtained by AFM and SEM showed the presence
of particles around 10-300 nm, and a good morphology for the films produced by mixing. The
colloids solutions showed particles with hydrodynamic ray (Rh) in the range 220–3000 nm,
and zeta potential values in the range 24–25 mV, exhibiting a moderate stability. The zeta
potential of chitosans and chitosan particles was around 25 mV.The minimum inhibitory
concentration (MIC), minimum bacterial concentration (MBC), in vitro antibacterial activity
and the integrity of cell membrane were investigated against Escherichia coli. The
antimicrobial studies showed that smallest particles have a high antimicrobial activity
presenting similar concentrations for MIC and MBC (0.2 mg/mL) upon the tested bacteria
(E. coli).
Keywords: Chitosan, Ultrassonication, Nanoparticles, Biofilms, Antimicrobial activity.
SUMÁRIO
1
INTRODUÇÃO..........................................................................................................18
2
REVISÃO BIBLIOGRÁFICA.................................................................................19
2.1
CAPITULO I
CHITOSAN
NANOPARTICLES:
PRODUCTION
BY
GREEN
CHEMISTRY,
PHYSICOCHEMICAL PROPERTIES AND APPLICATIONS IN FOOD PRESERVATION
(Submetido, à convite para International Journal of Polymer Science)..................................19
2.1
INTRODUÇÃO...........................................................................................................20
2.1.1 Quitosana....................................................................................................................23
2.1.2 Bioatividade da quitosana.........................................................................................25
2.1.2.1
Atividade antimicrobiana da quitosana............................................................26
2.1.2.2
Grau de desacetilação da quitosana..................................................................26
2.1.2.3
Peso molecular.................................................................................................27
2.1.2.4
Efeito do pH.....................................................................................................28
2.1.2.5
Propriedade de solubilidade.............................................................................28
2.1.2.6
Derivatização....................................................................................................29
2.1.2.7
Temperatura e armazenamento........................................................................29
2.1.2.8
Espécies microbianas (Microbial species).......................................................29
2.1.3 O uso da nanotecnologia na produção de quitosana...............................................29
2.1.4 Ultra-som aplicado a quitosana................................................................................32
2.1.5 Avaliação da actividade antimicrobiana da quitosana...........................................34
2.1.6 Aplicação da actividade antimicrobiana na indústria de alimentos......................36
2.1.6.1
As culturas alimentares....................................................................................37
2.1.6.2
Preservação de alimentos.................................................................................38
2.1.6.3
Tecnologia de Alimentos.................................................................................38
2.1.6.4
Embalagens ativas para alimentos....................................................................39
2.1.6.5
Nanotecnologia em alimentos..........................................................................40
2.2
CONCLUSÃO ............................................................................................................41
3
CAPITULO II
PROPRIEDADES DE MECANICAS DE FILMES E SOLUÇÕES POLIMERICAS
3.1
REOLOGIA................................................................................................................46
3.3.1
Classificação dos Fluidos..........................................................................................46
3.3.2
Reologia aplicada a quitosana..................................................................................50
3.3.3 Comportamento de polímeros em solução...............................................................51
3.3.4
Aplicação na formação de filmes.............................................................................52
3.3.4.1. Propriedades mecânicas dos filmes............................................................................54
3.3.4.2 Propriedade de barreira...............................................................................................55
3.3.5
Biofilmes de quitosana..............................................................................................56
4
OBJETIVOS..............................................................................................................57
4.1
OBJETIVOS GERAL..................................................................................................57
4.2
OBJETIVOS ESPECÍFICOS.......................................................................................57
5
CAPITULO III – ESTUDO ORIGINAL 1..............................................................58
TWEAKING THE MECHANICAL AND STRUCTURAL PROPERTIES OF
COLLOIDAL CHITOSANS BY SONICATION (Publicado em Food Hydrocolloids)
5.1
INTRODUÇÃO...........................................................................................................59
5.2
EXPERIMENTAL.......................................................................................................61
5.2.1 Materiais e soluções...................................................................................................61
5.2.2 Degradação do biopolímero e análise por DLS.......................................................62
5.2.3 Preparação das misturas e medidas reológicas.......................................................63
5.2.4 Microscopia de força atômica...................................................................................63
5.3
RESULTADOS E DISCUSSÃO................................................................................64
5.3.1 Estudo por DLS..........................................................................................................64
5.3.2 Testes mecânicos sob cisalhamento dinâmico.........................................................66
5.3.3 Medidas de cisalhamento estacionário.....................................................................68
5.3.4 Regra Cox-Merz e efeito da temperatura................................................................71
5.3.5 Estudo por AFM.........................................................................................................72
5.4.
CONCLUSÃO............................................................................................................76
6
CAPITULO IV– ESTUDO ORIGINAL 2...............................................................95
FABRICATION AND CHARACTERIZATION OF FIBERS CHITOSAN-BASED
FILMS REINFORCED WITH CHITOSAN NANOPARTICLES (manuscrito em
desenvolvimento)
6.1
INTRODUÇÃO...........................................................................................................96
6.2
MATERIAL E MÉTODOS.........................................................................................97
6.2.1 Materiais e soluções...................................................................................................97
6.2.2 Ruptura das fibras de quitosana por ultrassonicação............................................98
6.4.3 Mistura das soluções..................................................................................................98
6.4.4 Produção dos filmes bioplásticos..............................................................................98
6.4.5 Cor e opacidade..........................................................................................................99
6.2.6 Microscopia eletrônica de varredura.....................................................................100
6.2.7 Características mecânicas.......................................................................................100
6.2.8 Permeabilidade ao vapor d’água............................................................................101
6.2.9 Isotermas de sorção a água.....................................................................................101
6.2.10 Analises estatísticas..................................................................................................102
6.3
RESULTADOS E DISCUSSÃO..............................................................................102
6.3.1 Propriedades mecânicas..........................................................................................102
6.3.2 Permeabilidade ao vapor d’água............................................................................107
6.3.3 Morfologia dos filmes de quitosana........................................................................113
6.3.4 Propriedades de cor....................................................................................................116
6.4
CONCLUSÃO...........................................................................................................118
7
CAPITULO V – ESTUDO ORIGINAL 3.............................................................120
ANTIMICROBIAL ACTIVITY OF CHITOSAN NANOPARTICLES OBTAINED BY
ULTRASOUND IRRADIATION.
7.1
INTRDUÇÃO............................................................................................................120
7.2
MATERIAL E MÉTODOS.......................................................................................122
7.2.1 Degradação do polímero..........................................................................................122
7.2.2 Espalhamento de luz dinâmico...............................................................................123
7.2.3 Potencial zeta............................................................................................................123
7.2.4 Atividade antimicrobiana........................................................................................123
7.2.4.1 Determinação da concentração inibitória mínima (MIC).........................................123
7.2.4.2 Determinação da concentração bacteriana mínima (MBC)......................................124
7.2.4.3 Inibição do crescimento pela quitosana in vivo........................................................124
7.2.5 Integridade da membrana celular..........................................................................125
7.3
RESULTADOS E DISCUSSÃO..............................................................................125
7.3.1
DLS e potencial zeta.................................................................................................125
7.3.2 MIC e MBC...............................................................................................................126
7.3.3 Integridade da membrana celualar.............................................................................127
7. 4
CONCLUSÃO...........................................................................................................130
8
DISCUSSÃO.............................................................................................................131
9
CONCLUSÕES........................................................................................................142
10
PESPECTIVAS FUTURAS....................................................................................143
11
REFERÊNCIAS.......................................................................................................144
LISTA DE FIGURAS
CAPITULO I
1
Estrutura molecular da (a) quitina e (b) quitosana (SHUKLA, SUDHEESH K. et
al., 2013) .................................................................................................................................42
2
Dados obtidos do website sciencedirect.com – Junho/2015-21:00 h, pesquisa
realizada usando as palavras: “Chitosan and Application”. .......................................43
3
As principais aplicações de quitosano. Fonte: sciencedirect.com – Junho/201521:00, pesquisa realizada usando as palavras: “Chitosan” and “Application”, entre
2000 e 2015. ................................................................................................................44
CAPITULO II
1
Classificação
dos
fluidos
segundo
seus
comportamentos
reológicos.
http://www.setor1.com.br/analises/reologia/cla_ssi.htm.............................................47
2
Representação do comportamento do fluido na Lei das Potencias. Curvas de fluxo
de fluidos da potencia, (1) fluidos dilatantes, (2) Newtonianos, (3) Pseudoplásticos
(Ostwald Waale), (4) Bingham e (5) Plástico (Carsson e Herschel . and Bulkley)
(BAXTER; ZIVANOVIC; WEISS, 2005) ..................................................................48
3
Fluxo entra placas paralelas......................................................................................48
4
Representação das fases de uma solução polimérica..............................................52
5
Curva de Tensão vs Deformação (VASYUKOVA et al., 2001). .............................54
CAPITULO III – ESTUDO ORIGINAL 1
1
a) As distribuições da amplitude do espalhamento de luz dispersa em função do
raio hidrodinâmico das partículas para CHIT90 sonicada para: (b) evolução da
intensidade de luz dispersa em cada um dos picos demonstrados em (a).............82
2
Espectros mecânicos..................................................................................................83
3
Evolução da tan como uma função da frequência oscilatória (ω) para as
amostras isoladas NS e S tratadas por 5, 15 e 30 min.............................................85
4
Curva de cisalhamento estacionário obtido para amostras pura NS e S (tS) 5 (a),
15 (b), and 30 min (c) ................................................................................................86
5
Comparação entre A , *e ’ medidas a 25ºC para as amostras de quitosana
NS e S (tS) ...................................................................................................................88
6
Imagens de AFM obtidas no modo tapping para substrato de mica recém clivada
recoberta com filmes finos de diferentes proporções de misturas coloidais NS:S
(tS) ................................................................................................................................89
CAPITULO IV– ESTUDO ORIGINAL 2
1
Efeito das diferentes proporções de misturas NS/S(ts) sobre diferentes atividades
d’água dos filmes, ajustados ao modelo de GAB...................................................111
2
Imagens representativas de SEM das secções transversais dos filmes na
magnificação de 30,000x de quitosana e nanaoparticulas....................................113
3
Imagens representativas de SEM das secções transversais dos filmes na
magnificação de 30,000x das misturas NS/S(ts) na proporção de 1:1.................114
CAPITULO V – ESTUDO ORIGINAL 3
1
Liberação de ácidos nucleicos e % de inibição de células de E. coli....................129
LISTA DE TABELAS
CAPITULO I
1
Aplicações de quitosana e seus derivados na indústria de alimentos....................45
CAPITULO II
1
Demonstração de modelos matemáticos aplicáveis a fluidos não-Newtonianos
(BAXTER et al., 2005) ...............................................................................................49
1
Dados de raio hidrodinâmico e amplitude obtidos da degradação da solução de
CHIT90.......................................................................................................................90
2
Parâmetros obtidos dos ajustes dos modelos de Cross e Carreaus a partir das
analises de cisalhamento oscilatório.........................................................................91
3
Dados de rigidez de superfície e tamanho médio extraídos das imagens obtidas
por AFM......................................................................................................................92
CAPITULO IV– ESTUDO ORIGINAL 2
1
Medias de espessuras (d) propriedades de tensão (resistência à tração, TS;
alongamento de ruptura, E; e modulo de Young, Y) e permeabilidade ao vapor
d’água (WVP) para filmes de quitosana preparados com Chito90 e misturas de
nanopartículas..........................................................................................................106
2
Parâmetros obtidos a partir do ajuste ao modelo de GAB..................................109
3
Parâmetros de cor....................................................................................................117
1
Caracterização das nanopartículas por DLS, raio hidrodinâmico (Rh),
intensidade do espalhamento de luz (% Intensity), índice de polidispersabilidade
(% PdI) e potencial zeta...........................................................................................125
2
MIC e MBC obtidos de diferentes amostras de quitosana contra E. coli...........128
LISTA DE ANEXOS
ANEXO I
Subimissão da revisão para International Journal of Polymer Science - CHITOSAN
NANOPARTICLES: PRODUCTION BY GREEN CHEMISTRY,
PHYSICOCHEMICALPROPERTIES AND APPLICATIONS IN FOOD PRESERVATION
ANEXO II
Artigo para Food Hydrocolloids - TWEAKING THE MECHANICAL AND
STRUCTURAL PROPERTIES OF COLLOIDAL CHITOSANS BY SONICATION
18
1 INTRODUÇÃO
Com o avanço do conceito da química verde que busca formas mais ecológicas e
menos agressivas ao meio ambiente na síntese e produção de macromoléculas vem sendo
propostas. A utilização de biopolímeros pode ser uma boa alternativa na substituição de
derivados do petróleo. A quitosana é uma macromolécula oriunda do processo de
desacetilação da quitina, que é o segundo polissacarídeo mais abundante na natureza. A
conversão da quitina em quitosana confere a essa molécula características que se
enquadram no conceito de química verde, incluindo características como serem atóxicas
biodegradáveis, biocompatíveis e apresentarem potencial antimicrobiano natural. Na
ultima década vem crescendo de forma exponencial o número de trabalhos que
apresentam diferentes aplicações para esses polímeros. A indústria de alimentos vem
enfrentando problemas com as embalagens convencionais, tais como plásticos e seus
derivados que são eficientes na conservação de alimentos, porém causam grande impacto
ao meio ambiente e adicionam um custo significativo ao produto final. A quitosana
aparece como opção na substituição desses materiais devido a suas características já
destacadas e a possibilidade de produção da mesma a partir de rejeitos da indústria
pesqueira (quitina oriunda do exoesqueleto de crustáceos). Outro ponto importante na
produção de biofilmes comestíveis a base de quitosana é sua alta atividade antimicrobiana
natural contra microrganismos patogênicos e deteriorantes, incluindo fungos e bactérias
Gram negativas e positivas. Porém os biopolímeros apresentar propriedades mecânicas e
de barreira relativamente pobres, o que atualmente limitam a sua utilização industrial. Em
especial a sua baixa propriedade de barreira a humidade devido à sua natureza hidrofílica.
No entanto sido implementada a ideia de que essas propriedades podem ser melhoradas
através da utilização de nanocompósitos. A formação de um nanocompósito para melhorar
as propriedades térmicas, mecânicas e de barreira tem provado ser uma alternativa
promissora. Com isso a utilização de macromoléculas biológicas como a quitosana como
base de embalagens bioativas vem se tormando um modo promissor e eficaz da
manutenção da qualidade dos produtos alimentícios.
19
2
REVISÃO BIBLIOGRÁFICA
2.1
CAPITULO I
CHITOSAN
NANOPARTICLES:
PHYSICOCHEMICAL
PRODUCTION
PROPERTIES
AND
BY
GREEN
CHEMISTRY,
APPLICATIONS
IN
FOOD
PRESERVATION (Submetido para International Journal of Polymer Science)
Laidson Paes Gomes, Vânia M. F. Paschoalin and Eduardo Mere Del Aguila*
Universidade Federal do Rio de Janeiro, Instituto de Química - Av. Athos da Silveira 149,
Cidade Universitária – 21949-909, Rio de Janeiro, RJ - Brazil.
*Corresponding author
Mailing address:
Instituto de Química, Universidade Federal do Rio de Janeiro
Av. Athos da Silveira Ramos 149 – Centro de Tecnologia – Bloco A - sala 545
Cidade Universitária - 21941-909
Rio de Janeiro, RJ, Brazil
Phone: +55 21 39887362
Fax: +55 21 39887266
E-mail: [email protected]
20
Abstract
Biologically active biomolecules such as chitosan and its derivatives have significant potential
in the food industry in view of contaminations associated with food products and the
increasing concerns in relation with the negative environmental impact of conventional
packaging materials. Chitosan is a biopolymer derivative of chitin, obtained from the waste of
industrial fishing activities. Its unique chemical structure as a linear polycation with high
charge density, reactive hydroxyl and amino groups, as well as extensive hydrogen bonding,
confer a wide range of applications to this compound. It is economical and easily available
and offers real potential for applications in the food industry due to its particular physicochemical properties, short time biodegradability, biocompatibility with human tissues,
antimicrobial and antifungal activities. These characteristics produce versatile substance that
give this biopolymer high potential for applications in food preservation. Meanwhile, food
nanotechnology as a new technology is in need of reviews regarding potentially adverse
effects, as well as its many positive effects. In this review, we aim to cover some of the
developments in chitosan nanotechnology by green chemistry and its applicability to the food
industry using its natural antimicrobial activity due to variations in polymer molecular weight
and degree of deacetylation.
Keywords: nanoparticles, chitosan, antimicrobial activity, coating, food preservation
2.1
INTRODUCTION
In the last years, the sustainable development concept has obtained important politic
and social attention due to the application of “green technologies” and the use of “green
products”, that contribute to sustainability through the decreasing of environmental
degradation, by establishing the best ways to synthesize and obtain products necessary for the
development of different areas, while at the same time reducing impacts on the environment
(DAHL; MADDUX; HUTCHISON, 2007).
Green chemistry explores chemistry techniques and methodologies that reduce or
eliminate the use or generation of feedstock, products, by-products, solvents, reagents, etc.,
that are hazardous to human health or to the environment (RAVEENDRAN; FU; WALLEN,
2003).
21
Solvent-free organic reactions are an important part of green chemistry and make it
possible to reduce the consumption of solvents unfriendly to the environment and allow for
the use of scaled down reaction vessels. Avoiding a certain solvent reduces the number of
components in a reaction, stops any solvent emission problems, and circumvents any solvent
recycling requirements. It is known that the best solvent is no solvent, but, if a solvent is
needed, then water is usually recommended, and catalysis in aqueous biphasic systems is an
industrially attractive methodology, which has found broad applications (SHELDON, 2005).
Two decades ago, however, a new approach to polymer synthesis has been developed,
employing enzymes as catalysts (enzymatic polymerization) for a review, see (KOBAYASHI;
UYAMA; KIMURA, 2001). In vitro enzymatic catalysis has been extensively used in the
organic synthesis area as a convenient and powerful tool (JONES, 1986;
WONG;
WHITESIDES, 1994) showing several advantages, including high efficiency, recyclability,
the ability to operate under mild conditions, and environmental friendliness (BAXTER et al.,
2005).
Natural biopolymers are attractive for different applications in improving human
health, aiding in drug or vaccine production or even in the production of food preservatives
and additives, since these compounds have inherently biocompatible and biodegradable
structures, are safe, and also are more easily accepted by health surveillance regulatory and
inspection institutions.
All biopolymers are produced in living cells by enzymatic catalysis. Research on
finding new enzymes and on the mechanisms of enzymatic reactions have been among the
most important central topics in fields such as biochemistry, organic chemistry, medicinal
chemistry, pharmaceutical chemistry and polymer chemistry (BRUICE, 2002).
Currently, many thousands of enzymes are commercially available and some of them
have suffered modifications for industrial applications. Generally, oxidoreductases,
hydrolases and isomerases are relatively stable, and some isolated enzymes among these are
conveniently used as catalysts in a practical manner in chemical, food and pharmaceutical
industrial plants.
All in vivo enzymatic reactions involve the following characteristics: (i) high catalytic
activity (high turnover number), (ii) reactions under mild conditions with regard to
temperature, pressure, solvent, pH of the medium, etc., bringing about energetic efficiency,
and (iii) high reaction selectivity of regio-, enantio-, chemo-, and stereo regulations, giving
rise to perfectly structure-controlled products. If these in vivo characteristics could be
22
obtained for in vitro enzymatic polymer synthesis, we may expect the following outcomes: (a)
perfect control of polymer structures, (b) creation of polymers with new structures, (c) a clean
and selective process without forming byproducts, (d) a low loading process that saves
energy, and (e) biodegradable properties of product polymers in many cases. These are
indicative of “green” nature of in vitro enzymatic catalysis for developing new polymeric
materials (KOBAYASHI; MAKINO, 2009), and in the polymer area the interest regarding
green chemistry is increasing.
Chitin is the second most abundant natural polymer in the world, after cellulose. It is
comprised of N-acetilglycosamines units bound by β-(1→4) linkages. The first in vitro
synthesis of chitin was accomplished in 1995 via ring-opening polyaddition of a chitobiose
oxazoline derivative monomer (a disaccharide) catalyzed by chitinase (EC 3.2.1.14), a
glycoside hydrolase of chitin working in its reverse reaction (KOBAYASHI; SAKAMOTO;
KIMURA, 2001; SATO, H. et al., 1998).
Chitin is found in three different crystalline forms; α, β and γ, dependent on the
arrangement of the individual chitin chains. In α-chitin, the most common chitin form, the
chains are arranged in an antiparallel way, and are densely packed with both inter- and
intramolecular hydrogen bonding, which explains the inability of α-chitin to swell in water
and also why α-chitin is the most rigid chitin form (BAXTER et al., 2005; CARREAU, 1972;
SOUSA et al., 2013). In β-chitin, the chains are packed in a parallel way (BORGES;
CAMPIÑA; SILVA, 2013;
BORMAN, 1990), resulting in a looser packing which can
incorporate small molecules into the crystal lattice to form various crystalline complexes. The
high degree of hydration and reduced packaging tightness give more flexible and soft
chitinous structures, as can be seen when comparing insect cuticles composed of β- versus αchitin. γ -chitin, the third and most controversial form of chitin, consists of two parallel
strands which alternate with a single antiparallel strand.
Commercial chitin can be found with various average degrees of acetylation (DA)
ranging from the fully acetylated to the totally deacetylated products. When containing higher
degrees of acetylation, this polymer is soluble in very few solvents, which limits its
application. Production is usually performed in heterogeneous conditions, and, because of this
then, the residual acetyl substituent distribution depends on the source of chitin, on the
conditions of deacetylation and on the degree of residual acetylation. It is clear that the
solubility of these polymers directly depends on the average degree of acetylation but also on
the distribution of the acetyl groups along the chains (BRUGNEROTTO et al., 2001).
23
The main application of chitin is involved with its structural modification, forming
chitosan by the deacetylation process. The derivative biopolymer Chitosan (CS) is formed,
when the deacetylation of chitin units is ≥60%. This compound shows higher solubility than
chitin and can, after glycosidic bond hydrolysis, form olygossacharids with variable molecular
weights.
2.1.1 Chitosan (CS)
Chitosan is primarily produced from chitin, which is widely distributed in nature,
mainly as the structural component of arthropod exoskeletons (including crustaceans and
insects). Structurally, chitin is an insoluble linear mucopolysaccharide (Fig. 1) consisting of
repeated N-acetyl-D-glucosamine (GlcNAc) units linked by β-(1→4) glycosidic bonds (fig.
1). The totally deacetylated chitosan molecule structure has an –NH2 group and two –OH in
each glycosidic residue, showing a polycationic character formed by –NH3+ radicals when in
pH solutions inferior to its pKa, with –NH3+ radicals (SHUKLA, SUDHEESH K. et al.,
2013). The relative amount of these two monomers (2- amine and 2- acetamide) can be
modified between the chitosans, depending on the extraction and production methods and the
organisms used for the extraction. The production of polymers with different physicalchemical characteristics, due to the degree of deacetylation (DD), is a consequence of the
amount of deacetylated radicals (2-amine) present in the sample, varying between 60 and
95%.
The depolymerization of chitosan molecules forms oligomers with variable amounts of
2-amino and 2-acetoamido units, producing low molecular weight chitosan (LMWCS)
ranging from 5000 to 10000 Da, which changes viscosity and pKa, among other parameters
(KITTUR; VISHU KUMAR; THARANATHAN, 2003).
CS is insoluble in both, organic solvents or water. It can, however, be readily
dissolved in weak acidic solutions, due to the presence of amino groups. The solubilization
occurs by protonation of the –NH2 on the C-2 position of the D-glucosamine repeat unit,
when the polysaccharide is converted to a polyelectrolyte in acidic media (SCHRAMM,
1994). To obtain a soluble product, the deacetylation degree (DD) of CS should reach 80–
85% or higher (KRAJEWSKA, 2005).
The removal of the acetate radical from the chitin molecule monomers can be carried
out by different methods, such as chemical hydrolysis, often performed using acids such as
24
HCl, nitrous acid, phosphoric acid, and hydrofluoric acid, or alkaline treatments or enzymatic
deacetylation (GOMES, LAIDSON P. et al., 2014).
The conversion of chitin to chitosan conducted by acid treatment is not used due to
the breakage susceptibility of the glycoside bonds of the polymer, since oligomers obtained in
his way are not bioactive, due to molecule destructuration
and the possibility of
environmental contamination and the creation of toxic chemical compounds (KIM, S.-K.;
RAJAPAKSE, 2005).
The most common method for chitosan synthesis is the deacetylation of chitin using
NaOH, which requires chitin exposure to severe temperature and NaOH concentration
conditions. This process has significant yield, but higher degrees of deacetylation require
more severe treatment conditions. To obtain chitosans with a degree of deacetylation of 85 to
92%, chitin should be exposed to harsh alkaline conditions, which can degrade. At the same
time, there is large consumption of water and energy, as well as impacts on the environment
due the large volume of discarded solvents (TAN et al., 2015).
Enzymes are used for an increasing range of applications. In fact, enzyme-catalyzed
processes have been gradually replacing chemical processes in many areas of the industry,
because they save energy, water and chemicals, help improve product quality, and,
furthermore, present valuable environmental benefits. These benefits are becoming more
important at a time of increasing awareness regarding sustainable development, green
chemistry, climate change and organic production (KANG; LIANG; MA, 2014). Different
hydrolytic enzymes able to catalyze the cleavage of glycoside bonds in chitosan have been
isolated (AZIZ et al., 2012; LECETA et al., 2013). Cellulase, pectinase, pepsin, papain,
neutral protease, lipase, and α-amylase show the ability to hydrolyze chitosans, at comparable
activity levels but with different specificities (GOMES, LAIDSON P.
et al., 2014;
MUZZARELLI, R. A. A.; TOMASETTI; ILARI, 1994; RONCAL et al., 2007).
These enzymes are obtained from different organisms and their use for the
deacetylation of chitin has been explored in order to improve process efficiency and control
over the physicochemical characteristics of the formed products. These enzymes are capable
of reducing chitin crystallinity and assay conditions can be adjusted to produce chitosan
macromolecules with different molecular weights (MW) between 4.0-10.0 kDa. Moreover, if
enzymes able to promote deacetylation of 2-acetamido monomers are associated to this
process, chitosans presenting different molecular weights and different degrees of
25
deacetylation can be obtained (DEL AGUILA et al., 2012; GOMES, LAIDSON P. et al.,
2014)
When the enzymatic conversion is compared to the commonly used chemical method,
in addition to environmental impacts, such as higher power consumption and the generation
of large amounts of toxic waste, the products formed during in the alkaline hydrolysis method
are heterogeneous, with variable MW and DA (TSIGOS et al., 2000).
The homogeneity in size and degree of deacetylation of the formed chitosans is very
important for the subsequent application of the generated product, and the higher the product
uniformity, better market value will be, since they may be used in several different types of
applications (BEANEY; LIZARDI-MENDOZA; HEALY, 2005).
The growing number of scientific papers and patents regarding chitosan and its
applications demonstrates a surprisingly high level of research on this biopolymer, both in
basic and in applied science.
The growth in the number of publications related to applications of chitosan in
different subject areas in the last 15 years is exponential: in early 2015, 5475 publications
were available, approximately twice the number of publications observed for 2009 (2848).
Eleven years ago, not even one thousand publications were available per year (838),
demonstrating the exponential increase of the study and importance of this biopolymer (fig.
2).
Areas of particular application regarding chitosan in the past 15 years are the
pharmaceutical industry, such as tissue engineering and drug transport, of 20 and 21%,
respectively. However, the versatility of chitosan application can be demonstrated by the
diversity of uses in many areas, ranging from purification of water contaminated with metal
ions to the formation of nanotubes and use as an antimicrobial agent. This versatility
contributes directly to the exponential growth of studies conducted with this macromolecule,
which is intrinsically related to new applications of this biopolymer (fig . 3).
2.1.2 Chitosan bioactivities
Many biological activities have been reported for CS, such as antimicrobial, anticancer,
antioxidant, and immunostimulant effects that are dependent on its physico-chemical
properties (Table 1). Food applications have already been approved by the Food and Drug
Administration in Japan, Italy and Finland (KEAN; THANOU, 2010). Chitosan has not been
26
officially proclaimed a GRAS by the FDA in United States for use in food, although this
regulatory agency has approved chitosan for medical uses, such as bandages and drug
encapsulation.
2.1.2.1
Antimicrobial activity of chitosan
Among the different CS bioactivities, perhaps the most applicable in the food
production chain is its antimicrobial activity, and this will be the focus of this review. The
antimicrobial activity of CS is influenced by several factors, which can be classified into four
categories (a) microbial factors related to the species of the target organism and cell age; (b)
intrinsic factors such as the density of positive charges, molecular weight, concentration,
hydrophilic/hydrophobic characteristics and chelating potential of chitosan; (c) physical state
and water solubility and (d) environmental factors, such as ionic strength of the medium, pH,
temperature and time of exposure to the pathogen (KONG et al., 2010).
2.1.2.2
Degree of deacetylation (DD) of chitosan
The DD is the great importance because chemical behaviors, such as solubility and
acid-base behavior, of both chitin and chitosan, depends considerably on this characteristic
(SORLIER et al., 2001). The DD is a parameter defined by routine analyses performed for
quality control on chitin and chitosan preparations and is essential for studying their chemical
structure, properties, and structure property relationships. If the DD is known, many
properties can be predicted.
Several spectroscopic methods are able to determine the DD, with the most usual
being X-ray diffraction (ZHANG, Y. et al., 2005), infrared spectroscopy (KASAAI,
MOHAMMAD R., 2008) and nuclear magnetic resonance (solid or liquid–state)
(KUMIRSKA et al., 2010).
The antimicrobial activity of chitosan is directly proportional to its DD (LIU, X. D. et
al., 2001). Increases in the DD cause an increase in the number of amine groups in the
chitosan macromolecule with an increase in the number of amino protonated groups (NH3+) in
acid media and complete dissolution in water, which results in the higher possibility of
interaction between chitosan and the cell walls of negatively charged microorganisms (S.
SEKIGUCHI; Y. MIURA; H. KANEKO, 1994). Generally, yeasts and fungi are chitosansensitive microorganisms, followed by Gram-positive bacteria, and, finally Gram-negative
27
bacteria (ING et al., 2012). The antimicrobial activity is higher at pH 6.0 (chitosan pKa = 6.2)
when a higher number of amine groups are in the form of free bases when compared to pH 7.5
(KENDRA; HADWIGER, 1984). Other studies tested chitosans with different DD, 92.5 %
and 85 %, and observed that polymers with higher deacetylation (high DD) were more
effective in controlling microorganism growth (SIMPSON et al., 1997).
2.1.2.3
Molecular Weight – MW
Molecular weight is also of fundamental importance; generally chitosans with lower
molecular weights and lower degrees of deacetylation exhibit greater solubility and faster
degradation than their high molecular weight counterparts. Detailed characteristics of chitosan
for biomedical applications are well described in a comprehensive review, where, due to the
presence of the reactive functional group, chitosans were modified into various forms, such as
thiolated, carboxyalkyl, bile acid-modified, quaternized (N, N, N-trimethyl chitosan; TMC),
sugar-bearing and cyclodextrinlinked chitosans (COTTER; HILL; ROSS, 2005) The main
methods used to determine the molecular weight of chitin and chitosan are viscosimetry
(KASAAI, MOHAMMAD R., 2007), light scattering (SORLIER et al., 2003), gel permeation
chromatography (GOMES, LAIDSON P.
et al., 2014), osmometry and sedimentation
equilibrium by ultracentrifugation (CÖLFEN; BERTH; DAUTZENBERG, 2001).
It has been shown that low molecular weight chitosans (LMW -CS ) of less than 10
kDa have greater antimicrobial activity compared to high molecular weight chitosans (CS HMW ) ranging between 10 and 500 kDa (UCHIDA; M. IZUME; A. OHTAKARA, 1989).
Studies with hydrochloride D-glucosamine (a chitosan monomer), however, were not
effective in inhibiting bacterial growth. This suggests that the antimicrobial activity of
chitosan is related not only to the cationic nature of the deacetylated glucosamine, but also
with the chain length of the biopolymer (BLAGODATSKIKH et al., 2013).
Although some results about the bactericidal activity of low molecular weight
chitosans are comparable, it has been reported that, depending on the bacteria strain, the
conditions of the biological assays and the physico-chemical characteristics (MW and DD) of
the chitosan in question, the results are not always in agreement. Studies with a 9.3 kDa
chitosan have shown this macromolecule to inhibit E. coli growth, while a 2.2. kDa chitosan
promotes the growth of this same bacteria (TOKURA et al., 1997). On the other hand, a 4.6
kDa chitosan was most active against Gram positive bacteria, yeast and fungi (TIKHONOV et
28
al., 2006). Thus, results with LMW-CS are still somewhat controversial and unclear,
indicating that more studies are required on the inhibition mechanisms of LHW-CS.
2.1.2.4
Effect of pH
It has been shown that the antimicrobial activity of chitosan and chitosan-based films
increases in lower pH. One theory postulates that the positive charge of the amine group
(NH3+) at lower pH values than the pKa (pH<6.3), at which this functional group carries 50%
of its total electric charge, allows for interactions with negatively charged microbial cell
membranes, a phenomenon which may cause leakage of intracellular constituent
(HELANDER et al., 2001). However, even if this is a well recognized explanation regarding
the antimicrobial effects of chitosan, there is no direct evidence demonstrating this behavior
against bacteria.
2.1.2.5
Solubility properties
Although
chitin
and
chitosan
have
been
confirmed
as
being
attractive
biomacromolecules with relevant antimicrobial properties, their applications are somewhat
limited due to both being water-insoluble. Water-soluble chitosan derivatives can be obtained
by the introduction of permanent positive charges in the polymer chains, resulting in a
cationic polyelectrolyte characteristic independent of the pH of the aqueous medium. This can
be accomplished, for example, by the quaternization of the nitrogen atoms of the amino
groups. According to Xie et al. 2002 (XIE et al., 2002), at neutral pH, the degree of
protonation of NH2 is very low, so the repulsion of NH3+ is weak. Under such conditions, the
intermolecular and intramolecular hydrogen bonds result in a hydrophobic micro-area in the
polymer chain, creating hydrophobic and hydrophilic portions, favoring the structural affinity
between the bacteria cell wall and the derivative. In another study, the cell supernatants of S.
aureus exposed to a water-soluble chitosan derivative in deionized water suggested that the
water-soluble chitosan produced by the Maillard reaction may be a promising commercial
substitute for acid-soluble chitosans (CHUNG; YEH; TSAI, 2011).
29
2.1.2.6
Derivatization
Chitosan-derivatives are usually obtained by chemical modifications of the amino or
hydroxyl (especially at the C6 position in the chitosan backbone) groups, in order to improve
their physicochemical properties (MARTINS et al., 2013). Some authors report that the
bactericidal activity of chitosan-derivatives is stronger than that of unmodified chitosan. Jia et
al (2001)(JIA; SHEN; XU, 2001) demonstrated that the N-propyl-N,N-dimethyl chitosan
presents a bactericidal activity against E. coli 20 times higher than that of chitosan with 96%
deacetylation, but with different MWs. Other authors reported that the antimicrobial activity
of N,N,N-trimethyl chitosan (TMC) is ca. 500 times higher than that of unmodified chitosan.
It has also been reported that other chitosan-derivatives, such as hydroxypropyl chitosan, Ohydroxyethylchitosan and carboxymethyl chitosan, among others, also exhibit significant
antimicrobial activity (JIA et al., 2001; SUN et al., 2014).
2.1.2.7
Temperature and Storage
For commercial applications, it would be practical to prepare chitosan solutions in
bulk and store them for further use. During storage, specific chitosan, viscosity or MW
characteristics might be altered. Therefore, the altered viscosity of a chitosan solution must be
monitored, since it may influence other functional properties of the solution. The stability of
chitosan solutions (MW of 2025 and 1110 kDa) and their antibacterial activity against Grampositive (Listeria monocytogenes and S. aureus) and Gram-negative (Salmonella enteritidis
and E. coli) bacteria were investigated at 4 °C and 25 °C after 15-week storage (NO et al.,
2006).
2.1.2.8
Microbial species
Chitosan exerts an antifungal effect by suppressing sporulation and spore germination
(HERNÁNDEZ-LAUZARDO et al., 2008). In contrast, antibacterial activity is a complicated
process that differs between Gram-positive and Gram-negative bacteria due to different cell
surface characteristics. . In several studies, stronger antibacterial activity was apparent against
Gram-negative bacteria when compared to Gram-positive bacteria (CHUNG et al., 2004),
while in another study Gram-positive bacteria were more susceptible, perhaps as a
consequence of the Gram-negative outer membrane barrier (ZHONG et al., 2008).
30
2.1.3 The use of nanotechnology in chitosan production
Nanoscience is an emerging field that deals with interactions between molecules, cells
and engineered substances, such as molecular fragments, atoms and molecules. In terms of
size constraints, the National Nanotechnology Initiative (NNI) defines nanotechnology in
dimensions of roughly 1 to 100 nanometers (nm) (TOMASIK; ZARANYIKA, 1995), but in a
broader range this can be extended up to 1000 nm. Particles found in this range appear to be
optimal for achieving a number of important tasks, such as nano-carriers, including alterations
of a drug’s reactivity, strength, electrical properties, and, ultimately, its behavior in vivo.
There is great interest in developing new nanodelivery systems for drugs that are already on
the market, especially cancer therapeutics (CHO, K. et al., 2008).
The major goals in designing nanoparticles (NP) as a delivery system are to control
particle size, surface properties and the release of pharmacologically active agents in order to
achieve the site-specific action of the drug at the therapeutically optimal rate and dose
regimen.
Nanoparticles can be prepared from a variety of materials such as proteins,
polysaccharides and synthetic polymers. The application potential of a nanoparticle depends
on certain factors, such as type of material (REN et al., 2009), particle shape (WANG, R. H.;
XIN; TAO, 2005) and concentration (KIM, B. et al., 2003). The intrinsic properties of
nanoparticles are determined predominantly, by their size, composition, crystallinity and
morphology, as mentioned previously (NADANATHANGAM VIGNESHWARAN et al.,
2006). The chemical composition of NP, their surface shape, charge, hydrophobicity, size
(NEL et al., 2006) and the presence or absence of functional groups or other chemical
compounds (MAGREZ et al., 2006) are important features that define the future applications
of these compounds.
Nanoparticles have been most frequently prepared by three methods: (a) dispersion of
preformed polymers; (b) polymerization of monomers; and (c) ionic gelation or coacervation
of hydrophilic polymers. However, other methods, such as supercritical fluid technology
(REVERCHON; ADAMI, 2006) and particle replication in non-wetting templates (PRINT)
(ROLLAND et al., 2005) have also been described in the literature for application in the
production of nanoparticles. The latter was claimed to maintain absolute control of particle
size, shape and composition, which could set an example for the future mass production of
nanoparticles in the industry (ASHOKKUMAR; GRIESER, 1999).
31
Over time various applications have been developed using nanoparticles prepared
from either synthetic polymers or natural polymers. Among natural polymers, chitosans are
widely explored for their biomedical and pharmaceutical applications (OCHEKPE;
OLORUNFEMI; NGWULUKA, 2009).
CS has been explored as a material of choice to form nanoparticles for the last decade.
Chitosan properties have been enhanced by making them nanoparticles. The unique character
of NP, such as small size and quantum size effect could result in chitosan nanoparticles
(CSNP). They are simple and inexpensive to manufacture, can be scaled-up in the production
process, and have unique sizes and large surface-to-volume ratio. They are mucoadhesive and
hydrophyllic in nature and, because of this; they provide good protection to encapsulated
drugs, increasing their clearance time and stability in the body.
Although CS alone is considered safe for oral administration, its properties may
change completely upon chemical modifications. Moreover, it is well-known that the
pharmacokinetic properties of a drug or excipient change considerably when included in a
nanoparticulate system. Thus, the in vivo fate is decided by the size, charge and surface
modifications of the NPs. This, in turn, can alter the toxicity profile of the NP itself, as these
properties influence the way the NPs interact with different types of cells, thus modifying
their cellular uptake, absorption through the gastrointestinal tract, tissue distribution and
excretion. This is the reason that the generally recognized as safe (GRAS) status of CS does
not apply for nanoparticulate formulations and depends primarily upon the conditions of
intended use (KEAN; THANOU, 2010).
In addition, the molecular weight and degree of deacetylation of chitosan also affect its
pharmacokinetic properties and toxicity. Thus, each and every derivative should be assessed
individually, both in free form and in nanoparticulate form. A recently published review by
Kean and Thanou in 2010 (KEAN; THANOU, 2010) has summarized all the current findings
related to CS toxicity.
Different methods have been used to prepare chitosan nanoparticles (CSNP), and the
selection of the method depends on factors such as particle size requirement, thermal and
chemical stability of the active agent, reproducibility of the release kinetic profiles, stability of
the final product and residual toxicity associated with the final product (AZEVEDO et al.,
2013). The main chitosan nanoparticles preparation methods are emulsion cross linking,
emulsion- droplet coalescence, coacervation/ precipitation, ionotropic gelation, reverse
micelles, template polymerization and self-assembly polyelectrolytes (BASTOS et al., 2010).
32
However, the selection of any of these methods depends on the nature of the active molecule,
as well as the type of the delivery device.
From a technical point of view, it is extremely important that chitosan be hydro
soluble and positively charged. These properties enable this polymer to interact with
negatively charged polymers, macromolecules and even with certain polyanions upon contact
in aqueous environment (PATEL, J.; JIVANI, 2009).
Even though chitosan nanoparticles appear to be safe in laboratory-scale studies, the
knowledge of the risks involved in real-world applications leaves much to be desired, since
there are some cases in which the use of chitosan nanoparticles has been questionable
(KUZMA; ROMANCHEK; KOKOTOVICH, 2008). However, the use of these particles on
the macroscale is questionable, as nanomaterials exhibit novel properties due to their
extremely small size, high surface area, and reactivity (MELO, 2008; OBERDÖRSTER;
OBERDÖRSTER; OBERDÖRSTER, 2005). Therefore, the consequences of releasing them
into the environment must be assessed.
The early findings of these particles in favor of human use at the nanolevel are
promising signs for their possible safe environmental applications. However, some doubts
have been aroused with regard to the use of nanoencapsulated food additives and nanocoated
films in food packaging (PERERA; RAJAPAKSE, 2014). However, complete toxicity effects
have not yet been studied and more exposure assessments are required in order to obtain a
better picture of the relationship between nanoparticle applications and their health risks.
Most of the time, the risks regarding nanoparticles are assessed with their chemical
composition and, to date, no widely accepted or well-defined risk assessment methods or test
strategies exist explicitly designed for nanoparticles (CONG et al., 2011). It is, thus, important
to gather more information regarding health and environmental risks associated with
nanoparticle applications, in order to identify the proper risk assessment strategies and
implement regulatory policies to ensure the safety of these nanomaterials.
2.1.4 Ultrasound applied to chitosan
In the polymer area, there is also increasing interest in green chemistry, in order to
design chemical products and processes that reduce or eliminate the use or generation of
hazardous substances, particularly if nanoparticles will be used in human beings. Correlation
between structure-property and structure-activity are often employed in the production, such
as enzymatic desacetylation and other non-classical activation methods developed in
33
accordance with ‘green chemistry’ requirements. Ultrasonic irradiation offers important
potential for the conversion of biomass raw materials such as polymeric carbohydrates to
useful lower weight molecules (GOMES, LAIDSON P. et al., 2014; KANG et al., 2014;
SAVITRI et al., 2014) .
The application of ultrasound is one of the most economical and simple tools for the
degradation of long polymeric macromolecules. Through temperature, frequency and intensity
control and polymer concentrations, the extent of the degradation is mainly determined by the
sonication time (ts) (BAXTER et al., 2005;
KASAAI, MOHAMMAD R; ARUL;
CHARLET, 2008). The scission of the polymeric chains for production the polymers with
molecular mass within a ts-dependent distribution can be produced in this way.
The ultrassonication technique was applied by Gallant et al 1972 (SOUSA et al., 2013)
and Degrois et al 1974 (DEGROIS et al., 1974), who were the first one to study the ability of
this technique in degrading starch granules. Ultrasonication is a common tool for the
preparation and processing of polymer nanoparticles. It is particularly effective in breaking up
aggregates and in reducing the size and polydispersity of nanoparticles (BERNE; PECORA,
1976). The physical stability and in vivo distribution of nanoparticles are affected by their
mean size, polydispersity, and surface charge density (BODMEIER; MAINCENT, 1998).
Despite the wide-spread applications of ultrasonication in nanotechnology, its effects on
chitosan nanoparticles are not well understood, although there have been several reports on
the ultrasonication of chitosan polymers.
High-intensity ultrasonication produces acoustic cavitation, which generates shortlived hot spots with intense local heating of 5000 °C, pressures of 1000 atm, and heating and
cooling rates above 1010 K/s (SUSLICK; PRICE, 1999). These, along with free-radical
formation, may mediate redox reactions and intramolecular regroupings in samples
(ELPINER, 1964). Cavitation also generates rapid streaming of solvent molecules around the
cavitation bubble, as well as shock waves during bubble collapse, which in turn generate very
large shear forces (TANG, E. S. K.; HUANG; LIM, 2003).
Recent studies have further investigated the effects that ultrasound treatments can
cause in polymer macromolecules, changing properties such as chemical composition, size,
shape, surface charge density, hydrophobicity (NEL et al., 2006), polydispersity and the
presence or absence of functional groups or other chemical agents (MAGREZ et al., 2006)
when subjected to different treatment conditions (IZIDORO et al., 2011). Other studies have
demonstrated that it is possible to improve the solubility, swelling power and the the capacity
of water absorption of starch samples after ultrasound treatment (24W with 40 % amplitude
34
and a frequency of 20kHz) (SOUZA, H. K. S. et al., 2013). The characterization of nano
products obtained by ultrasonication is extremely important to determine their possible
applications.
Several methods have been developed for the production of chitosan nanospheres,
such as ionotropic gelation, spray drying , emulsification, coacervation, and more recently,
ultrasonication (SINHA et al., 2004). The development of stable chitosan nanospheres is
limited by the ionic interaction between chitosan and the anionic compound, which may be an
alginate, tripolyphosphate (TPP) , carrageenan or polyelectrolyte (KIM, S. et al., 2013).
Most importantly, previous evidence on the ultrasonic degradation of chitosan
supports that scission occurs mainly through the β-(1→4) linkage and that DD remains barely
unaffected even for long sonication times (BAXTER et al., 2005; LI, J.; CAI; FAN, 2008).
2.1.5 Evaluation of the antimicrobian activity of chitosan
The antimicrobial potential of chitosan has been observed in different developmental
stages in fungi, such as micelial growth, sporulation, spore viability and germination and the
production of virulence factors. The antimicrobial activity of chitosan is further associated to
physico-chemical characteristics (molecular mass, deacetylation degree and solution pH), also
depends on the target organism and its developmental phase (DINA RAAFAT et al., 2008;
TSAI; SU, 1999).
The natural antimicrobial properties of chitosan and its derivatives have resulted in
their extensive use as commercial disinfectants, since some chitosans and derivatives have an
advantage over other types of disinfectants due to their high antimicrobial activity, broad
spectrum of activity and low toxicity to mammalian cells, allowing for their discard with less
damage to the environment and its derivatives (LU et al., 2013a).
It has been shown that the difference between the antimicrobial potential of chitosanbased films and their derivatives may be directly involved with variations in particle size, film
thickness and the structure of the matrix-forming fibers. Another study determined the
structure and particle size of two different films, with particles between 74-500 μm
(resembling a flake), and particles between 37-63 μm (resembling a sphere) where the
antimicrobial activity of the film against S. aureus was higher in the film with smalled-sized,
spherical shaped particles, which provides greater specific surface (TAKAHASHIA et al.,
2008)
35
Some studies describe the CS mechanism of action as a result of the interaction
between the chitosan macromolecules, that are positively charged, and the membranes of the
microbial cells, that are negatively charged, with breakage occurring and, consequently,
leakage of intracellular components, including proteins (JUNG et al., 1999; LIU, H. et al.,
2004). The CS interacts with the cell membrane, altering its permeability (LEUBA;
STOSSEL, 1986) and also acting as a chelating agent selectively binding to trace metals, thus
inhibiting the production of toxins and microbial growth. CS also activates various defense
processes in the host tissue (EL GHAOUTH et al., 1992), acting as a binding agent to water
and as an inhibitor of several enzymes.
Another described mechanism of inhibition is the interaction between the positively
charged chitosan with the cellular DNA of fungi and bacteria, which consequently inhibits the
RNA and protein syntheses. This mechanism occurs only with low molecular weight
chitosans (LMW-CS) which can penetrate microorganism cells. This mechanism of action is
still controversial, but could explain the inhibition of Gram-positive and Gram-negative
bacteria and fungi, establishing a similar mechanism of action for all, regardless of cell
membrane structure, due to ionic interactions with DNA (SUDARSHAN, N. R.; HOOVER,
D. G.; KNORR, D., 1992).
On the other hand, the mechanism for antibacterial activity appears to be different
when considering Gram-positive and Gram-negative bacteria, due to the different
characteristics of each type of cell membranes. Still, many researchers have demonstrated that
there were no significant differences between antibacterial activities (WANG, X.; DU; LIU,
2004). Based on the available evidence, it seems that bacteria in general are less sensitive to
the antimicrobial activity of chitosan than fungi, since their growth occurs at pH ≤ 6.0
(ROLLER; COVILL, 1999) It is believed that the observed differences are attributed to the
characteristics of each of the studied microorganisms, mainly with regard to the growth phase
of the cell, since the charge of the microbial cell surface varies depending on this parameter
(BAYER; SLOYER, 1990; TSAI; SU, 1999).
Chitosan molecular weight and deacetylation degree may contribute together to the
solubility of this biopolymer, thus explaining seemingly controversial results. The
modification of the molecular weight changes the content of N-acethylglucosamine units in
chitosan, which have both an intramolecular and intermolecular influence, resulting in
chitosans with different conformations. However, increasing chitosan solubility implies in
control of the deacetylation of the residues, which is sometimes a low yield process
(KURITA; KAMIYA; NISHIMURA, 1991).
36
Chitosan definitely demonstrates a strong inhibitory effect on microorganism the
growth at low pHs, since this antimicrobial activity is weakened with increasing pH (KONG
et al., 2008), due to the low protonation of amino groups, which also influences in decreasing
the solubility of the biopolymer (AIEDEH; TAHA, 2001; PAPINEAU et al., 1991).
The different physical states and molecular weight of chitosan and its derivatives
provide distinct mechanisms of antibacterial action. Low molecular weight chitosans and
water-soluble ultrafine nanoparticles can penetrate bacteria cell walls, binding with DNA and
inhibition of mRNA synthesis that occurs through chitosan penetration in microorganism
nuclei and interference with mRNA and protein synthesis (SUDARSHAN, N. R. et al., 1992).
High molecular weight and water soluble chitosans and solid chitosans, including larger
nanoparticles, in turn interact with the cell surface and alter cell permeability (LEUBA;
STOSSEL, 1986), resulting in or forming an impervious layer around the cell, thereby
blocking the transport of essential solutes into the cell (CHOI et al.; EATON et al., 2008).
2.1.6 Application of antimicrobial activity in the food industry
It is accepted that chitosan nanoparticle-based films can be effectively used in the food
industry, as they provide various benefits, including good edibility, biocompatibility with
human tissues, an aesthetically pleasing appearance, barrier properties against pathogenic
microorganisms, atoxicity non-polluting, and low cost (GAVHANE; GURAV; YADAV,
2013). Their applications are listed in the table 1.
Food companies are looking for strategies to make their products healthier without
compromising quality and consumer perception. Biopolymer nanoparticles are being studied
for their ability to protect and target the delivery of bioactive ingredients, and/or to design
foods with novel physicochemical or sensory attributes. An important area of current research
is the identification of new sources of biopolymers and new types of preparation methods that
can be used to fabricate biopolymer particles with novel or improved functional attributes.
However, it is also important that the ingredients and processing methods used be
economically feasible for large-scale production. In addition, the biopolymer particles
produced must be compatible with food and beverage matrices, must protect encapsulated
ingredients during food storage, and must release them in an active form at the desired site of
action.
37
2.1.6.1
Food crops
Chitosan can be used primarily as a natural seed treatment and plant growth enhancer,
and as an ecologically friendly biopesticide substance that boosts the innate ability of plants to
defend themselves against fungal infections (EL-SAWY et al., 2010). Chitosan applications
for plants and crops are regulated by the EPA, and the USDA National Organic Program
regulates its use on organic certified farms and crops (LINDEN et al., 2000). EPA-approved,
biodegradable chitosan products are allowed for use outdoors and indoors on plants and crops
grown commercially and by consumers (USDA; EPA, 2007).
Chitosan has prevented numerous pre- and postharvest diseases on various
horticultural commodities. Microscopical observations indicate that chitosan had a direct
effect on the morphology of chitosan-treated microorganisms reflecting its fungistatic or
fungicidal potential. In addition to a direct microbial activity, other studies strongly suggested
that chitosan induced a series of defense reactions correlated with enzymatic activities
(KATIYAR et al., 2014).
The foliar application of chitosan in pepper plants decreased transpiration and reduced
water use by 26-43%, while maintaining biomass production and yield. Hence, chitosan might
be an effective antitranspirant to conserve water use in agriculture (BITTELLI et al., 2001;
HIRANO et al., 1990) reported that coating seeds with depolymerized chitosan or its
oligosaccharides typically increased chitinase activity in seedlings by 30-50%, unless the
seeds had a hard cuticle. A low molecular weight chitosan (5 kDa) induced the accumulation
of phytoalexins in plant tissue, decreased the total content and changed the composition of
free sterols, producing adverse effects on infesters, activating chitinase, beta-glucanase,
lipoxygenases and stimulating the generation of reactive oxygen species (VASYUKOVA et
al., 2001).
Chitosan had been shown to increase the production of glucanohydrolases, phenolic
compounds and the synthesis of specific phytoalexins with antifungal activity, and also
reduces macerating enzymes such as polygalacturonases, pectin metil esterase etc (YANG, Y.
et al., 2000) studied maize seeds and reported that chitosan concentrations at 2-4 g/litre
resulted in positive effects on endogenous hormone content, alpha-amylase activity and
chlorophyll content in seedling leaves.
In addition, Chitosan also induced structural barriers, for example, inducing the
synthesis of a lignin-like material. For some horticultural and ornamental commodities,
chitosan increased harvested yield, due to its ability to form a semipermeable coating, and
38
extends the shelf life of treated fruit and vegetables by minimizing the rate of respiration and
reducing water loss. Other studies showed that chitosan at 0.1 or 0.5% increased leaf area, leaf
dry weight and leaf length of soybean, lettuce and rice, whereas chitosan at 0.1% showed
positive effects on leaf area, leaf length and dry weight of tomato (CHIBU, 2000). As a
nontoxic biodegradable material, as well as an elicitor, chitosan has the potential to become a
new class of plant protectant, assisting towards the goal of sustainable agriculture
(HERNÁNDEZ-LAUZARDO et al., 2008).
2.1.6.2
Food preservation
Coatings based on chitosan were used as an antifungal agent and in enhancing the
germination and quality of artichoke seeds. The effect of the formulation and thickness on
seed germination (G%), fungi activity and vegetative growth were studied by Ziani et al, 2009
(ZIANI et al., 2009). Results indicated significant differences between treatments on seed
germination and it was observed that all chitosans reduced the number of fungi strains and
increased plant growth.
In another study, apples (Malus domestica Borkh. cv. Gala) were heat-treated at 38°C
for 4 days (heat treatment) before or after being coated with 1% chitosan. The heat treatment
+ chitosan treated fruit showed the lowest respiration rate, malondialdehyde, membrane
leakage, ethylene evolution and the highest firmness and consumer acceptance among the
treatments (SHAO et al., 2012).
In other studies, when applied on wounded wheat leaves, chitosan induced
lignification and, consequently, restricted the growth of nonpathogenic fungi in wheat.
Chitosan also inhibited the growth of A. flavus and aflatoxin production in liquid cultures,
pre-harvest maize, and groundnut, and also enhanced phytoalexin production in germinating
peanut (CUERO; OSUJI; WASHINGTON, 1991). In addition, Chitosan also improved the
microbiological quality of fresh cut broccoli (MOREIRA; ROURA; PONCE, 2011).
2.1.6.3
Food technology
Chitosan films have shown potential to be used as a packaging material for the quality
preservation of a variety of foods. It has also been widely used in antimicrobial films to
provide edible protective coating, and in the dipping and spraying of food products due to its
antimicrobial properties.
39
In a review, Dutta et. al., 2009 (DUTTA et al., 2009) aimed to highlight various
preparative methods and antimicrobial activities including the mechanism of the antimicrobial
action of chitosan-based films. The optimisation of the biocidic properties of these so called
biocomposite films and the role of biocatalysts in the improvement of quality and shelf life of
foods has been discussed elsewhere. The use of chitosan-based edible films could be a
promising technique to preserve the microbial quality of pork meat hamburgers. Its
importance in the modulation of the oxygen permeability of films in order to avoid the
undesirable effects of metmyoglobin (MtMb) formation was studied by Vargas et al., 2011
(VARGAS; ALBORS; CHIRALT, 2011), promoted by the lower oxygen partial pressure in
the surface of the coated hamburgers. The addition of sunflower oil to chitosan-based films
yielded glossier films and led to an improvement in the water vapour barrier properties when
acetic acid was used as a solvent to prepare the films. Juice clarification had been successfully
carried out for apple, carrot, grape, lemon, orange and pineapple juices by Rungsardthong et
al. 2006 (RUNGSARDTHONG et al., 2006).
2.1.6.4
Active food packaging
Although conventional packaging materials, such as plastics, are effective for food
preservation, they have created many serious environmental problems. The use of
environmentally friendly, cost-effective packaging materials has gained considerable attention
from scientists, and the use of bio-based active films appears to be a better solution in respect
to these requirements (AIDER, 2010). The use of chitosan nanoparticle-based edible films has
been reported on many occasions (DE MOURA et al., 2011). The versatility of chitosan
nanoparticles has generated a wide range of applications in the food industry. The main focus
has been to develop chitosan-based films to be used as packaging material to act against
bacteria and fungi, to improve shelf life by preserving the food and maintaining food quality.
The interest in edible coatings is on the rise due to their ability to reduce fruit
respiration and transpiration rates, and increase storage time and consistency retention
(DEBEAUFORT; QUEZADA-GALLO; VOILLEY, 1998; VELICKOVA et al., 2013; VU
et al., 2011). In order to achieve this purpose, their potential non-toxic, biodegradable,
antimicrobial, low relative cost of production and the approval of its use as a food additive by
the “United States Food and Drug Administration” (USFDA) were tested (KNORR, 1986).
The use of chitosan decreased the respiration rate and production of ethylene in
raspberries (TEZOTTO-ULIANA et al., 2014), and has a great selective permeability to
40
respiratory gases, acting as a passing barrier for O2 (ELSABEE; ABDOU, 2013). This control
of gases between the fruit and the environment reduces respiration rates, 1-carboxylic-1aminocyclepropane oxidase and syntases enzyme action, which are highly influenced by the
presence O2 (COLINET et al., 2010). The reduction of mass loss with chitosan use was also
related to the formation of a selective barrier around the surface of the fruit, improving
moisture loss and reducing respiration and the main metabolic processes that lead to loss of
water (HAN et al., 2004; HONG et al., 2012).
Edible coatings consisting solely of chitosan or a combination of chitosan with other
biopolymers, such as sodium caseinate, were applied to carrots, cheese and salami
(MOREIRA, MARIA DEL ROSARIO et al., 2011). The sodium caseinate/chitosan films
inhibited bacteria and yeast and can be applied to different food matrices. In other studies,
acetic or propionic acid were incorporated into a chitosan matrix in bologna ham, baked ham
and fresh salmon (SCHIRMER et al., 2009).
The application of high concentrations of chitosan is considered effective in the
control of fruit colouring, decreasing fruit darkening and maintaining anthocyanin content, a
pigment directly related to food freshness. Enzyme activity is influenced by the presence of
O2 presence inside the fruit. As chitosan forma a barrier around the fruit, darkeningis reduced.
Furthermore, the positive charges present in the coating can stabilize anthocyanines, helping
in maintaining fruit colour (TEZOTTO-ULIANA et al., 2014).
The United States Department of Agriculture (RATKOVICH et al., 2013)
implemented a project to develop green nanotechnology for eliminating foodborne pathogens.
In this study, the USDA envisaged the development of a nanoparticle wash treatment with the
capability of significantly reducing or eliminating pathogenic bacteria associated with fresh or
fresh-cut fruits and vegetables, to be used with minimal processing. The specific tasks involve
the design, synthesis, and characterization of ultrapotent chitosan nanoparticles coated by
antimicrobial peptides, the evaluation of peptide-enhanced nanoparticles as a lysis agent in
realistic food processing environments, and the development of a postharvest nanoparticle
electric field treatment for decreasing the bacterial load of fresh fruits and vegetables.
2.1.6.5
Food nanotechnology
The versatility of chitosan nanoparticles has generated a wide range of applications in
the food industry. The main focus was to develop chitosan-based films to be used as
packaging material to act against bacteria and fungi and to improve shelf life by preserving
41
the foodstuff and maintaining food quality. The use of chitosan nanoparticle- based edible
films as a food coating has been reported with respect to a variety of food, including cheese
and meat products, such as fermented sausages (WANG, X. et al., 2004). In another study,
Sato et al. (2012) (SATO, A. C. K. et al., 2011) demonstrated the possibility of producing
food-grade stable nanoparticles with simple processing techniques, using lecithin and sodium
caseinate, which could be further used as base systems for the production of nanocapsules.
2.2
CONCLUSIONS
Chitosan is a versatile food biopolymer that has a variety of applications in all areas of food
science. Chitosan possesses promising broad spectrum antimicrobial activities, and has, thus,
been widely studied as a food preservative to improve food quality and extend the shelf life of
perishable food products. All of these intrinsic properties vary with MW and DD, both of
which are the most important characteristics of chitosan. Chitosan is, therefore, a versatile and
promising biodegradable polymer for food packaging. In addition, it possesses immense
potential as an antimicrobial packaging material, due to its antimicrobial activity and nontoxicity.
Inherent antibacterial properties and its film-forming ability make chitosan an ideal
choice for use as a biodegradable antimicrobial packaging material that can be used to
improve the storability of perishable foods. It has convincingly been proved that chitosan
films exhibit good antimicrobial activity, which can help in extending food shelf life. Hence,
it is no surprise if a widespread use of chitosan films is witnessed in tomorrow’s food
packaging.
Acknowledgments
The authors acknowledge the financial support of Fundação Carlos Chagas Filho de Amparo à
Pesquisa do Estado do Rio de Janeiro (FAPERJ, Rio de Janeiro, Brazil), Conselho Nacional
de Desenvolvimento Científico e Tecnológico (CNPq, Brasília, Brazil) and Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brasília, Brazil).
42
Legends
Figure 1. Molecular structures of (a) chitin and (b) chitosan (SHUKLA, SUDHEESH K. et
al., 2013)
43
Figure 2 – Data obtained at the site sciencedirect.com – June/2015-21:00 h, search using the
following words: “Chitosan and Application”.
44
Chitosan Aplication
6%
8%
Drug delivery - 21%
21%
Tissue engeneering - 20%
Drug release - 10%
9%
Metal ion - 9%
Carbon nanotube - 9%
8%
20%
Delivery system - 8%
Anti bacterial - 9%
9%
Stem cell - 8%
9%
10%
DNA - 6%
Figure 3 – Major chitosan applications. Source: sciencedirect.com – June/2015-21:00, search
using the following words: “Chitosan” and “Application”, between 2000 and 2015.
45
Table 1. Applications of chitosan and its derivatives in the food industry (GAVHANE et
al., 2013).
Use
Antimicrobial agent
Example
Bactericidal, fungicidal; measure of
contamination in agricultural commodities
mold
References
(MARTÍNEZ-CAMACHO
et al., 2010; YANG, T.-C.;
CHOU; LI, 2005)
Edible film industry
Controlled moisture transfer between food and the
surrounding environment; controlled release of
antimicrobial substances, antioxidants, nutrients,;
reduction of partial oxygen pressure; controlled rate
of respiration, temperature control
(ABUGOCH et al., 2011; AI
et
al.,
2012;
DEVLIEGHERE;
VERMEULEN;
DEBEVERE,
2004;
KHUNAWATTANAKUL et
al., 2011)
Additives
Clarification and deacification of fruits and
beverages, natural flavor extender, texture
controlling agent, emulsifying agent, colour
stabilization
(ALBERTENGO
2013;
et
G,
al.,
2007;
RUNGSARDTHONG et al.,
2006)
Nutritional quality
Dietary fibre; hypocholesterolemic effect; livestock
and fish feed additive, reduction of lipid absorption;
production of single cell proteins; antigastritic agent
(CARREAU,
LINDEN
1972;
et
al.,
2000;
MAEZAKI et al., 1993)
Recovery of solid
materials from food
processing wastes
Affinity flocculation;
Fractionation of agar
(RENAULT et al., 2009;
ZENG; WU; KENNEDY,
2008)
Purification
potable water
of
Recovery of metal ions, pesticides phenols and
PCB´s; removal of dyes
(BORGES
et
al.,
BORMAN, 1990).
2013;
46
3
CAPITULO II
PROPRIEDADES MECANICAS DE FILMES E SOLUÇÕES POLIMÉRICAS
3.1
REOLOGIA
As analises reológicas levam em consideração os esforços internos que um dado
material é ou não capaz de suportar, e as suas correspondentes deformações durante a
aplicação de uma tensão. Portanto pode ser definida como a ciência que estuda como a
matéria se deforma ou escoa, quando está submetida a um esforço originado por forças
externas, podendo o material estar no estado líquido, gasoso ou sólido. Cada material possui
características reológicas especificas, embora existam alguns tipos básicos de comportamento
mecânico (elasticidade, plasticidade e viscosidade) que permitem analisar o comportamento
reológico de inúmeros materiais, tais como fluidos.
Por definição um fluido pode ser uma substancia que não possui forma própria e
assume o formato do recipiente. A resistência de um fluido contra qualquer mudança de
posição irreversível de seus elementos é chamado de viscosidade. Para que se mantenha um
fluxo em um fluido é necessário aplicação de uma força contínua sobre o mesmo
(BRUNETTI, 2008). Os fluidos são gases ou líquidos que apresentam pequenas diferenças
entre si. O gás ocupa todo o volume do recipiente, enquanto o líquido apresenta uma
superfície livre (JAYAKUMAR et al., 2010). Diferentemente da relação de sólidos e líquidos
quanto a respostas quando submetidos a tensões de deformação, gases e líquidos não
apresentam nenhuma diferença quanto suas respostas reológicas (SCHRAMM, 1994).
3.3.1
Classificação dos Fluidos
Os materiais quando apresentados na forma de fluidos são agrupados em diferentes
subclassificações, levando em consideração seu comportamento reológico (figura 1). Esses
fluidos são classificados, quanto à relação entre a taxa e a tensão de cisalhamento,
comportando-se como fluido Newtoniano (água, ar, mel, etc.) quando possuem
proporcionalidade constante entre a taxa de cisalhamento e tensão de cisalhamento, caso não
apresente esse comportamento são considerados fluidos não-Newtonianos apresentando uma
relação não linear. Havendo ainda uma subdivisão entre os fluidos não-Newtonianos
(viscoelásticos, dependentes pelo tempo e independentes do tempo) apresentada na figura 1.
47
Figura
1.
Classificação
dos
fluidos
segundo
seus
comportamentos
reológicos.
http://www.setor1.com.br/analises/reologia/cla_ssi.htm
O entendimento e o controle das propriedades reológicas são de fundamental
importância na fabricação e no manuseio de uma grande quantidade de materiais (PATEL, M.
P.; PATEL; PATEL, 2010) como plástico (CHUNG et al., 2004), alimentos (MOHANRAJ;
CHEN, 2007), cosméticos (MEGHA AGARWAL et al., 2015), tintas óleos (AGNIHOTRI;
MALLIKARJUNA; AMINABHAVI, 2004), lubrificantes (SOWJANYA et al., 2013) e em
processos de bombeamento de líquidos em tubulações e moldagem de plásticos (PITTS et al.,
2014).
Os parâmetros reológicos para cada fluido podem ser determinados através de
modelos matemáticos particulares para cada fluido, o qual influência diretamente na
interpretação dos resultados de respostas do fluido. Os modelos mais comumente utilizados
são os de Newton, de Ostwald de Waale (Lei das potências), de Bingham e de Herschel and
Bulkley apresentadas na tabela 1. A figura 2 mostra as curvas de fluxo características
referente aos modelos matemáticos mencionados acima.
A viscosidade dos fluidos Newtonianos pode ser descrita matematicamente através da
experiência realizada por Newton (figura 3), onde o fluido cisalhado entre uma placa móvel e
uma placa estacionaria gera a equação (1), a qual descreve o comportamento de fluidos ideais.
𝜏 = 𝜂𝛾̇
equação 1
Onde 𝜏 representa a tensão de cisalhamento, força por unidade de área cisalhante expresso em
N/m2 ou pascal (Pa), 𝜂 é o valor da viscosidade, expresso em Pa·s e 𝛾̇ é a variação da
deformação (taxa de cisalhamento) em função do tempo.
48
Figura 2. Representação do comportamento do fluido na Lei das Potencias. Curvas de fluxo de fluidos da
potencia, (1) fluidos dilatantes, (2) Newtonianos, (3) Pseudoplásticos (Ostwald Waale), (4) Bingham e (5)
Plástico (Carsson e Herschel . and Bulkley) (RATKOVICH et al., 2013).
Em geral os polímeros são moléculas que geram fluidos com comportamento nãoNewtoniano, devido sua capacidade de formar géis de diferentes resistências ao fluxo,
dependendo da estrutura e concentração. Como a lei de Newton não se aplica a esses fluidos
existem diferentes modelos empíricos que são propostos para se calcular a viscosidade
aparente desses fluidos (tabela 1). A escolha do modelo a ser aplicado depende das
características do fluido, sendo a utilização do modelo correto de fundamental importância
para a obtenção de resultados que expressem o real comportamento de resposta do fluido.
Figura 3. Fluxo entra placas paralelas.
49
Tabela 1. Modelos matemáticos aplicáveis a fluidos não-Newtonianos (RATKOVICH
et al., 2013):
Lei da Potância (Ostwald de Waele)
𝜏 = 𝜂𝑎 𝛾 𝑛
Bringham
𝜏 = 𝜏0 + 𝑘𝛾̇
Herschel and Bulkely (0<n< ∞)
𝜏 = 𝜏0 + 𝑘𝛾̇ 𝑛
Casson
𝜏 0,5 = 𝜏00,5 + 𝜇͚ 0,5 𝛾̇ 0,5
Sisko
𝜇 = 𝜇͚ + 𝑘𝛾̇ 𝑛−1
Cross
𝜇 − 𝜇͚
1
=
𝜇0 − 𝜇͚ 1 + (𝜆𝛾̇ )𝑚
Carreau
𝑛−1
𝜇 − 𝜇͚
= (1 + (𝜆𝛾̇ )2 ) 2
𝜇0 − 𝜇͚
O modelo de Ostwald-de-Waele conhecido como lei da potencia é bastante utilizado
para descrever comportamentos de fluidos. Durante experimentos em que visavam examinar o
comportamento de fluidos sob escoamento cisalhante, Ostwald verificou que estes
apresentavam comportamentos muito divergentes dos previstos por Newton, esses fluidos
apresentavam uma relação entre tensão de cisalhamento (𝜏) por taxa de deformação (𝛾̇ ) não
linear. Diante disso ele propôs o modelo matemático descrito na equação 2:
𝜏 = 𝐾 𝛾̇ 𝑛 ....................Equação 2
50
Onde 𝐾
representa o índice de consistência, o qual indica o grau de resistência do
fluido diante do escoamento e 𝑛 uma grandeza adimensional que representa o índice da lei das
potências do comportamento do fluido, indicando o afastamento físico do modelo
Newtoniano, quando 𝑛 > 1 o fluido é considerado dilatante (figura 2, linha 1), encontrado em
suspensões concentradas de amido, quando a viscosidade aparente e a taxa de deformação
crescem na mesma proporção, 𝑛=1 fluido Newtoniano, nesta condição 𝐾 é convertido para 𝜂
(figura 2, linha 2) e 𝑛 < 1 fluido pseudoplástico (figura 2, linha 3), em que a viscosidade
aparente decresce com a taxa de deformação. A maior parte dos fluidos não-newtonianos
apresentam este comportamento (MELO, 2008).
O entendimento do fenômeno reológico é baseado em duas teorias principais: (i)
teorias que usam relações macroscópicas tensão-deformação-tempo e (ii) teorias moleculares
que fornecem evidências de comportamento viscoelástico da estrutura e conformação
molecular. Para polímeros sintéticos, ambas as relações são validas, entretanto para sistemas
biológicos o que pode ser considerado uma dispersão heterogênea, exibe uma considerável
variabilidade nas propriedades reológicas devido à complexidade inerente da amostra, por
isso, a aplicação de modelos reológicos clássicos é limitada para certos tipos de amostra
(ZOON et al., 1990).
3.3.2
Reologia aplicada à quitosana
A reologia pode ser utilizada para definir, por exemplo, o comportamento do fluxo de
polímeros fundidos a fim de caracterizar a sua capacidade de processamento, dando
percepções da estrutura molecular, e indicando influências relacionadas com a mistura de
polímero ou composição.
A investigação do comportamento reológico das soluções de quitosana vem sendo
realizada por meio de estudos que avaliam a influência de diferentes parâmetros: tais como
composição química, concentração, força iônica, pH e o efeito do tipo de ácido, além do
envolvimento do fenômeno de associação hidrofóbica ligado ao comportamento viscoelástico
das soluções de quitosana (HAMDINE; HEUZEY; BÉGIN, 2005) Alguns modelos
matemáticos representam de forma mais clara as respostas do material, sendo que para
soluções de quitosana os modelos de Cross (CROSS, 1965) e Carreau (CARREAU, 1972)
tem representado de forma mais clara seu comportamento.
51
A quitosana vem sendo amplamente utilizada em estudos que tem com objetivo a
formação de filmes e géis através da mistura da mesma a moléculas com potencial reticulante
(SCHIFFMAN; SCHAUER, 2007). Em relação à formação de géis quimicamente reticulados
por meio de ligações covalente entre as cadeias com quitosana, ARGÜELLES-MONAL et al.
(1998) realizaram um estudo sobre a dinâmica oscilatória de sistemas apresentados na forma
de géis quitosana-glutaraldeido. Devido às características ligadas a viscoelasticidade, bem
como as propriedades mecânicas e a função das ligações cruzadas que formam os géis, os
autores sugeriram que a formação de um gel reticulado ocorre, devido ao comportamento das
moléculas de glutaraldeido, que forma uma ponte entre as cadeias de quitosana restringindo a
mobilidade das mesmas. Sistemas como esse podem apresentar várias formas no processo de
relaxação, na qual a água contida nas ligações das moléculas do sistema de gel podem estar
envolvidas com o processo de relaxação das ligações de hidrogênio (CHENG et al., 1998).
Estudos demonstraram propriedades reológicas e de inchamento em amostras de
quitosana-polietileno, onde foi relatado que essa mistura pode ter sua capacidade de
inchamento controlada pela modificação do pH do meio, e que ocorre a formação de uma rede
interpenetrante na presença do óxido de etileno o que faz aumentar suas propriedades elásticas
(KHALID et al., 1999). Essas propriedades reológicas apresentadas por hidrogéis
pseudoplasticos podem ser alteradas dependendo do tipo e teor das substâncias misturadas e
das interações que ocasionalmente irão ocorrer entre os polímeros e/ou as substâncias,
BODEK (2000) com amostras de quitosana/metilcelulose ligadas a fármacos.
3.3.3 Comportamento de polímeros em solução
As propriedades das soluções poliméricas não são apenas dependentes das
características físico-químicas dos polímeros, mas estão diretamente dependentes à sua
concentração (C). Quando em soluções diluídas, as cadeias poliméricas se organizam de
forma isolada umas das outras separadas pelo solvente, diminuindo a possibilidade de
agregação das mesmas, facilitando assim a determinação, do volume hidrodinâmico e da
conformação das moléculas. Estes parâmetros são de grande importância na determinação das
propriedades físicas dos polímeros (KASAAI, MOHAMMAD R et al., 2008). No regime
concentrado, as macromoléculas estão entrelaçadas e as dimensões das cadeias são
independentes da concentração polimérica. O regime diluído e o concentrado são definidos a
partir do overlap concentration (C*), que é a concentração de polímero na qual as cadeias
começam a se sobrepor e a entanglement concentration (Ce), concentração na qual acima dela
52
as cadeias formam emaranhados e são independentes da concentração do polímero (Figura 4).
Resumindo, a solução é considerada diluída quando C < C* e soluções concentradas quando
C > Ce (CHO, J. et al., 2006).
Figura 4. Representação das fases de uma solução polimérica.
Acima de C*, as propriedades reológicas das soluções poliméricas no estado
semidiluído e concentrado tem sido caracterizada em termos da constante cisalhamento
simples e da pequena amplitude oscilatória de cisalhamento. A regra de Cox-Merz (COX;
MERZ, 1958) vem sendo amplamente usada em polímeros fundidos, concentrados ou semi
diluídos para estimar a viscosidade de cisalhamento constante 𝜂(𝛾̇ ) a partir da viscosidade
dinâmica e complexa |𝜂∗ (𝜔)|, que pode ser mais facilmente obtido através da equação 3:
∗
𝜂(𝛾̇ )|𝛾=𝜔
̇ = |𝜂 (𝜔)|..............................equação 3
Essa regra foi aplicada com sucesso em grande número de soluções poliméricas (CARREAU;
DE KEE; CHHABRA, 1997; LAUTEN; NYSTRÖM, 2000). Esse princípio pode prever
𝜂(𝛾̇ ) a partir de medidas oscilatórias, como também pode ser prevista a |𝜂∗ (𝜔)| a partir de
dados obtidos da viscosidade estacionária (steady) como demonstrado na equação 3. Essa
estimativa pode sofre variações quando avaliada em alta frequência.
53
3.3.4
Aplicação na formação de filmes
A indústria de embalagens e de alimentos tem unido esforços para reduzir os custos
sobre a produção e sobre a quantidade de embalagens de alimentos descartados no meio
ambiente, introduzindo a utilização de materiais biodegradáveis, como alternativa favorável
ao meio ambiente, em substituição aos polímeros sintéticos. Muitos dos materiais plásticos
tradicionais, como poliestireno e polipropileno, não são biodegradáveis nem recicláveis,
gerando aumento de resíduos. De acordo com a Environmental Protection Agency (EPA),
mais de 13 mil toneladas de resíduos de embalagens plásticas foram gerados nos Estados
Unidos em 2010 (KAHHAT; WILLIAMS, 2012).
Como alternativa ao uso de filmes plásticos vem sendo introduzido à utilização de
filmes formados por biopolímeros, os quais são oriundos de recursos renováveis e geralmente
obtidos a partir de matérias primas brutas como celulose e proteínas; a partir da síntese de
monômeros bioderivados como o polilactato, ou ainda, de polímeros naturais produzidos por
microrganismos como polihidroxibutirato e polihidroxivalerato (PETERSEN et al., 1999).
Polímeros biodegradáveis podem ser utilizados na forma de filmes e/ou revestimentos
comestíveis. Os filmes comestíveis podem ser utilizados individualmente como cobertura de
pequenos produtos alimentares, que não são constantemente embalados individualmente, tais
como cerejas (YAMAN; BAYOΙNDΙRLΙ, 2002), pistache (JAVANMARD, 2008) e
cogumelos (EISSA, 2007). Outras aplicações de filmes comestíveis também podem ser
observadas em embalagens de sopas secas ou em bebidas em pó (KROCHTA; MULDERJOHNSTON, 1997). O interesse em coberturas comestíveis vem crescendo devido a sua
capacidade de reduzir as taxas de respiração e transpiração, e aumentar o período de
estocagem e a retenção de firmeza de frutos (VU et al., 2011). Além disso, filmes poliméricos
biodegradáveis podem ser usados como agente carreador de diversos tipos de aditivos.
Dentre alguns sistemas de cobertura, a quitosana vem sendo testada com vantagem
devido as suas características de atoxicidade, biodegradabilidade e potencial antimicrobiano,
incluindo o baixo custo relativo de produção e a aprovação do seu uso como aditivo alimentar
foi feito pelo “United States Food and Drug administration” (USFDA) (KNORR, 1986).
Com isso a aplicação desses materiais pode melhorar a qualidade dos produtos alimentares.
54
3.3.4.1.
Propriedades mecânicas dos filmes
A determinação das propriedades mecânicas dos biofilmes inclui a avaliação da
resistência à tração, alongamento, deformação e modulo de elasticidade que são de
fundamental importância para que o material tenha a força mecânica necessária para manter a
integridade do produto durante o manuseio e armazenamento.
A resistência à tração é definida como a máxima de tensão que o material pode
suportar e é considerada como sendo a carga máxima exercida sobre a amostra durante o
ensaio. O alongamento é a alteração máxima no comprimento do filme antes da ruptura e é
expressa como a percentagem de alteração do comprimento original do material. O modulo de
elasticidade (Modulo de Young) é definido como a razão entre a tensão exercida e a
deformação sofrida pelo material, representada pela parte linear inicial da curva de tensão
(MCHUGH; KROCHTA, 1994). A curva típica de ensaio de tração está representada na
figura 5.
Figura 5. Curva de tensão vs deformação, onde a curva de tensão real expressa o
comportamento de um filme polimérico (RESIN, 2010).
55
As propriedades mecânicas dos biofilmes dependem da composição e da condição
ambiental. A incorporação de plastificantes aumenta a mobilidade das cadeias poliméricas o
que leva ao aumento do alongamento e diminuição da resistência a tração (SOTHORNVIT, R.
et al., 2007). Já a incorporação de outros aditivos podem melhorar propriedades do filme. A
umidade no ambiente afeta diretamente as propriedades mecânicas dos filmes. Os filmes
hidrofílicos absorvem a umidade mais rapidamente, aumentando o efeito da água, que age
como plastificante entre as cadeias poliméricas, que por consequência diminui a resistência à
tração, aumentando a extensibilidade do filme e expondo o produto armazenado a uma
humidade excessiva (CHO, S. Y.; RHEE, 2002).
3.3.4.2
Propriedade de barreira
Uma parte significativa da perda de qualidade dos alimentos está relacionada com
fatores ligados à transferência de umidade, gases, aroma e cor, a partir de influências do
ambiente. Durante o período de armazenamento, diversas reações químicas e/ou enzimáticas
são deteriorantes ao alimento, tais como, oxidação lipídica, reação de Maillard e
escurecimento enzimático, bem como o crescimento de microrganismos deteriorantes que
ocorre com o aumento da à atividade de água um fator critico no armazenamento de produtos
alimentares. Embalagens produzidas por biopolímeros devem ter propriedades de
transferência de massa apropriadas, de maneira a manter o produto conservado. A perda de
umidade de frutas e legumes durante um longo período de armazenamento pode resultar em
perda de massa e encolhimento (OLIVAS; BARBOSA-CÁNOVAS, 2005). Da mesma forma
transferências indesejada de gases podem causar problemas, como no caso da oxidação de
óleos quando expostos a uma quantidade excessiva de oxigênio devido à difusão indesejada
do ambiente para o alimento, causando perda nutricional e deterioração da textura, sabor, cor
e aroma, causando eventual desvalorização do produto (MATE; KROCHTA, 1996).
A natureza e composição do biofilme e as condições ambientais (umidade, pressão e
temperatura) tem grande influência nas propriedades de barreira nos produtos alimentares.
Polímeros com elevada polaridade (filmes a base de amido, quitosana e proteínas) possuem
elevada permeabilidade ao vapor d’água, porém baixa permeabilidade a gases (MCHUGH;
KROCHTA, 1994).
56
3.3.5
Biofilmes de quitosana
O uso de quitosana como base na formação de filmes, tem a capacidade de diminuir as
taxas de respiração e a produção de etileno em framboesas (TEZOTTO-ULIANA et al.,
2014). A quitosana apresenta excelente permeabilidade seletiva aos gases respiratórios,
atuando como barreira da passagem de O2 (ELSABEE; ABDOU, 2013).
A redução da perda de massa com o uso da quitosana também foi relacionada com a
formação da barreira seletiva em torno da superfície do fruto, reduzindo: a perda de umidade
e a respiração, e os principais processos metabólicos que levam a perda de água (HONG et al.,
2012; SOTHORNVIT, RUNGSINEE; RHIM; HONG, 2009).
A adição de nanopartículas a matrizes poliméricas com o intuito de formar
nanocompósitos a fim de melhorar as características dos filmes poliméricos (propriedades
mecânicas, térmicas e propriedade de barreira) vem sendo amplamente explorada.
Nanocompósitos são materiais onde um dos seus constituintes apresenta dimensão menor do
que 100 nm (ARORA; PADUA, 2010). O TiO2 e o ZnO na forma de nanopartículas são
exemplos de nanopartículas que vem sendo explorados (SOTHORNVIT, RUNGSINEE et al.,
2009). A adição desses nanopartículas a uma matriz, a fim de formar um nanocompósito, é
capaz de altera significativamente as propriedades mecânicas, diminuir a permeabilidade ao
vapor d’água e melhorar o potencial antimicrobiano dos filmes contra patógenos alimentares.
Existe um grande número de estudos que examinam o efeito da quitina/quitosana,
caseinato de sódio/quitosana e ácidos orgânicos na utilização em películas para o controle do
crescimento de microrganismos, com aplicação na forma de embalagem de alimentos
(MOREIRA, MARIA DEL ROSARIO et al., 2011) Entretanto, pouco vem sendo estudado na
avaliação das formas nanoparticuladas dessas substâncias com aplicação para o mesmo fim.
O potencial de aplicação para uma dada nanopartícula depende de alguns fatores,
incluindo tipo do material (REN et al., 2009), forma da partícula (WANG, R. H. et al., 2005)
e concentração usada (KIM, B. et al., 2003). As propriedades intrínsecas das nanopartículas
são determinadas predominantemente a partir da forma, tamanho, composição, cristalinidade
e morfologia (NADANATHANGAM VIGNESHWARAN et al., 2006). A avaliação da
atividade antimicrobiana de quitosanas de baixo e médio peso molecular em comparação com
conservantes usuais da indústria alimentícia (ácido benzóico e ácido sórbico), concluiu que as
quitosanas apresentaram melhor atividade antimicrobiana são melhores conservantes
industriais (CRUZ-ROMERO; KERRY, 2011). As nanopartículas solúveis de quitosana tem
grande potencial na utilização como antimicrobiano em embalagens bioativas.
57
4
OBJETIVOS
4.1
OBJETIVO GERAL
Estabelecer uma nova metodologia para a produção de bioplásticos de nanopartículas
a partir de quitosana e avaliar as suas propriedades como filme.
Estudar a atividade antimicrobiana das nanopartículas de quitosana contra patógenos
alimentares.
4.2 OBJETIVOS ESPECÍFICOS
Produzir nanopartículas de quitosana de forma controlada, pelo processo de
ultrassonicação;
Avaliar as características mecânicas e estruturais das soluções, produzidas a partir de
diferentes proporções de misturas entre fibras/nanopartículas de quitosana, a partir de
diferentes métodos reológicos;
Produzir biofilmes de quitosana a partir de misturas entre fibras/nanopartículas de
quitosana;
Avaliar a influência das nanopartículas: na morfologia, nas propriedades mecânicas,
na permeabilidade a vapor de água e morfologia dos filmes produzidos;
Avaliar o potencial antimicrobiano das fibras e nanopartículas de quitosana sobre
patógenos alimentares;
58
3
TWEAKING
THE
MECHANICAL
AND
STRUCTURAL
PROPERTIES
OF
COLLOIDAL CHITOSANS BY SONICATION (Publicado na Food Hydrocolloids)
Laidson P. Gomes,1,2 Hiléia K. S. Souza,1* José M. Campiña,3* Cristina T. Andrade, 4 Vânia
M. Flosi Paschoalin,2 A. F. Silva,3 and Maria P. Gonçalves1
1
REQUIMTE/LAQV, Departamento de Engenharia Química. Faculdade de Engenharia. Universidade do Porto.
Rua Dr. Roberto Frias, 4200-465, Porto, Portugal.
2
LAABBM, Instituto de Química, Universidade Federal do Rio de Janeiro, UFRJ, Av. Athos da Silveira Ramos
149, CT, Bl A, sala 545, Ilha do Fundão, CEP 21941-909, Rio de Janeiro Brazil.
3
Centro de Investigação em Química da Universidade do Porto (CIQ-UP). Departamento de Química e
Bioquímica. Faculdade de Ciências. Rua do Campo Alegre, 687, 4169-007, Porto, Portugal.
4
Programa Ciência de Alimentos, Instituto de Macromoléculas Professora Eloisa Mano, Universidade Federal
do Rio de Janeiro, Centro de Tecnologia, Bloco J 21941-598, Rio de Janeiro, RJ, Brazil
Abstract
Compared to the oil-derived plastics typically used in food packaging, biofilms of pure
chitosan present serious moisture issues. The physical degradation of the polysaccharide with
ultrasound effectively reduces the water vapor permeability in these films but, unfortunately,
they also turn more brittle. Blending chitosans of different morphology and molecular mass
(M) is an unexplored strategy that could bring balance without the need of incorporating toxic
or non-biodegradable plasticizers. To this end, we prepared and characterized the mixtures of
a high-M chitosan with the products of its own ultrasonic fragmentation. Biopolymer
degradation was followed by dynamic light scattering (DLS) and the mechanical and
structural characteristics of the mixtures were evaluated from different rheological methods
and atomic force microscopy (AFM). The results indicate that, through the control of the
sonication time and mixture ratio, it is possible to adjust the viscoelasticity and morphological
aspect of the mixtures at intermediate levels relative to their individual components. In a more
general sense, it is emphasized the importance of design and materials processing for the
development of a novel generation of additive-free sustainable but functional bioplastics.
Keywords: Biofabrication, Ultrasound, Colloidal Chitosan, Rheology, AFM
59
5.1
INTRODUCTION
The growing societal demands on sustainability and environmental protection are
expected to trigger a deep transformation of industrial manufacturing. In this sense, the
harnessing of abundant biomass resources must pave the way towards a greener bioeconomy.
Natural biopolymers are suitable resources for biofabrication because of their good
biodegradability, biocompatibility, and abundance. Chitosan (a polysaccharide obtained by Ndeacetylation of chitin, its natural precursor) which is usually extracted from insect and
marine crustacean shells (DEL AGUILA et al., 2012) is widely used in the food and cosmetic
industries due to its good processability as hydrogels, membranes, colloidal microparticles,
nanoparticles, and so on (IKEDA et al., 1993; JAYAKUMAR et al., 2010; LAPLANTE;
TURGEON; PAQUIN, 2006; LIU, Z. et al., 2012; PAN et al., 2002; PILLAI; PAUL;
SHARMA, 2009; SCHRAMM, 1994; SHUKLA, SUDHEESH K. et al., 2013; TOMASIK;
ZARANYIKA, 1995). Due to its great versatility, the use of chitosans in a new generation of
nanobiomaterials for analysis (BORGES et al., 2013), catalysis, tissue engineering (KIM, I.Y. et al., 2008), gene-delivery, and other applications (MIZRAHY; PEER, 2012; PAYNE;
RAGHAVAN, 2007), is being widely investigated.
Regarding its use in biofabrication, applications have been mainly restricted to thin
films for food packaging and dressings for surgical wounds (MADHUMATHI et al., 2010;
SEBTI et al., 2007). Although the replacement of non-biodegradable plastics derived from
crude oil by materials based on renewable and more sustainable resources, such as natural
biopolymers, has been anticipated since the early 90s (BORMAN, 1990;
CHIELLINI;
SOLARO, 1996; HOSOKAWA et al., 1990), the attempts to reproduce 3D objects of pure
chitosan with the good organization (and structural properties) which usually characterize
chitin systems in nature, have failed so far. In this respect, a significant step forward has been
given very recently thanks to the development of a new manufacturing strategy that yields
chitosan items in a variety of colors and shapes and with good mechanical properties for
commercial exploitation (FERNANDEZ; INGBER, 2014). In the particular field of food
packaging, chitosan films have evidenced poorer mechanical properties and much higher
moisture sensitivity than their oil-derived counterparts (MIMA et al., 1983; NADARAJAH,
2005). Unfortunately, the large retention of water turns these films unsuitable for their use in
direct contact with foodstuffs (OLABARRIETA et al., 2001). To circumvent these issues,
researchers have explored using different solvents (NADARAJAH et al., 2006), chemically-
60
derivatized chitosans (NIKOLAEV et al., 1987), or blending chitosan with synthetic or
natural compounds (referred to as plasticizers) (ZIANI et al., 2008).
The compatibility, miscibility, morphology, and spinnability of chitosan blends have
been extensively assessed in the literature (AZEVEDO et al., 2013; FERNANDES et al.,
2009;
HOMAYONI; RAVANDI; VALIZADEH, 2009;
KUMAR et al., 2010;
RAO;
JOHNS, 2008; WRZYSZCZYNSKI et al., 1995). For instance, (FERNANDES et al., 2009)
found enhanced mechanical properties in chitosan/bacterial cellulose biofilms. (AZEVEDO et
al., 2013) reported a notable improvement of the elasticity in biofilms of chitosan/cellulose
blends. In general, the properties of these blends are mainly determined by the compatibility
between their components: i.e. whether inter- and intra-molecular interactions between the
polysaccharide and the plasticizer lead to an intimate combination and macroscopically
uniform physical properties (KUMAR et al., 2010). However, leaving apart the controversial
migration of the plasticizer into the packaged food, the brittle nature of these biomaterials
remains a challenge to overcome. Ultrasonic irradiation has emerged as a rapid, easy-to-use,
and low-cost tool for the degradation of biopolymers such as starch (TOMASIK;
ZARANYIKA, 1995), pectins (SESHADRI et al., 2003) etc. Whereas the chosen frequency
mostly determines the energy available to trigger a random or a bond-specific chain scission
(in chitosan it occurs, preferentially, through the -(14) linkage), the intensity and
irradiation time (tS) control the extension of the process (BAXTER et al., 2005;
CZECHOWSKA-BISKUP et al., 2005; KASAAI, MOHAMMAD R et al., 2008).
Engineering films made of pure natural and biodegradable components with improved
moisture resistance but still retaining good shear strength and flexibility, remains a challenge
to improve the sustainability of the food packaging industry. In a recent paper, we
demonstrated that the sonication of a chitosan with high degree of deacetylation (DD=90%)
and molecular mass (M) induces a transition from a material mostly composed of colloidal
microfibers (non-sonicated sample) to a morphology dominated by the presence of spherical
nanoparticles (NPs) whose size is reduced upon increasing tS (SOUZA, H. K. S. et al., 2013).
In parallel with this transition, the mechanical properties of the corresponding biofilms turned
increasingly poorer (with a significant decline of the tensile strength and Young´s modulus).
Interestingly, the lowest water permeabilities were exhibited by the films prepared from the
most degraded chitosans what suggests that the high surface areas and surface-to-volume
ratios characterizing the nanoparticulated systems may provide a physical barrier to water
molecules.
61
Under the light of these results, one may wonder whether using mixtures of fibrous and
nanoparticulated chitosans as film precursors would bring some balance by ensuring both
reasonable mechanical properties, thanks to the presence of the microfibers, and enhanced
moisture resistance potentially provided by the NPs (a graphical representation of the concept
is shown in Scheme 1). In this work, we evaluate these prospects by preparing colloidal
mixtures of high-M (non-sonicated) chitosans and their corresponding low-M counterparts
(obtained by ultrasonic irradiation) at different ratios. The viscoelastic behavior and
morphology of the mixtures have been investigated by means of rheological measurements
and atomic force microscopy (AFM).The dynamic light scattering technique (DLS) has been
used to estimate the size distributions of chitosan colloids before, NS, and after sonication for
different times, S (tS).
5.2
EXPERIMENTAL
5.2.1 Materials and Solutions
For this investigation, a commercial ChitoClear® chitosan kindly supplied by Primex
(Siglufjordur, Iceland) was used. This product is extracted from shrimp shells from the North
Atlantic Ocean further de-acetylated to a degree of DD=90%. Although the average M was
declared to fall in the range 250-300 kDa, the viscosity-average molecular mass (MV) is over
650 kDa as it was determined in a previous work (SOUZA, H. K. S. et al., 2013). Hereinafter,
we will refer to this high-M chitosan as CHIT90. Glacial acetic acid (Merck, purity: >99%),
sodium acetate trihydrate (Merck, suprapur), sodium chloride (NaCl, Sigma-Aldrich,
>99.9%), and ethanol (Aga), were all analytical grade and used without further purification. A
0.1 M acetate buffer solution pH 6.0 (ACB) was prepared in ultrapure water (18.2 MΩ s).
When required, the pH was adjusted by addition of drops of glacial acetic acid or sodium
acetate. CHIT90 was suspended at 3% (w/w) in this buffer and, after gently stirring for 2 h,
stored at 4 °C (stock suspension). Before any measurement or ultrasonic treatment, aliquots of
this stock were incubated at room temperature (RT) for 60 min. The equilibrated samples
were, then, centrifuged for 20 min at 21,000 x g (9 ACC, 20 °C) with a Beckman Coulter
centrifuge Alegra 25R to remove the large aggregates. Thereby, a stable colloidal dispersion
of CHIT90 was obtained.
62
5.2.2 Biopolymer Degradation & DLS Analysis
After centrifugation, 30 mg aliquots of the CHIT90 dispersion were placed in ice and
ultrasonicated for a period of time between 0 and 30 min increased at 2 min per sample (the
so called sonication time, tS). A SONIC ultrasonic probe (model 750 W), equipped with a 1/2"
tip, was used to this end (constant duty cycle and 40% amplitude). The ultrasonic degradation
of CHIT90 was followed by dynamic light scattering (DLS). For these purposes, aliquots of
the sonicated dispersions were diluted 100 times and placed in fluorescence quartz cuvettes
(Hellma 111-QS, 10 mm light path). The measurements were acquired at constant
temperature (20 ºC) in a high-performance W130i DLS system (Avid Nano, UK) equipped
with a 660 nm light source and operated at 90 º. In a previous viscosimetric study (SOUZA,
H. K. S. et al., 2013), it was shown that the progressive sonication of CHIT90, under identical
conditions to those applied in this work, results in a continuous decrease of the viscosityaverage molecular mass (MV) from a value of 660 kDa (non-sonicated sample) to 540 (tS=5
min), 327 (15 min), and 291 kDa (30 min).
Although DLS analysis is commonly applied to small spherical particles (BERNE;
PECORA, 1976), it also works with more complex samples containing species of different
size and morphology (e.g. polymers, native proteins and their various aggregates, etc)
(LORBER et al., 2012). In order to minimize uncontrolled external factors and multiple
scattering effects (which typically affect the analysis of concentrated or highly polydisperse
samples), 5 measurements were consecutively gathered for each sample and the results were
analyzed with the software i-Size 2.0. The average values of the diffusion constant (D) and
the hydrodynamic radius (Rh) were, thereby, extracted. The polydispersity index (PdI) was
also estimated by comparison of the averaged autocorrelation curve with the perfect
exponential of a monodisperse reference sample. From Rh and PdI, i-Size calculated the
particle size distributions (plotted in respect to Rh). The statistical significance was assessed
from the standard deviation of the different measurements collected for each sample. Other
relevant parameters, such as the % of intensity scattered by each type of particle (% intensity),
were also derived from this analysis (see Table 1).
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5.2.3 Mixture Preparation and Rheological Measurements
The colloidal mixtures were prepared by adding the proper weight of CHIT90 stock
dispersion (the non-sonicated component henceforth referred to as NS-CHIT90) to aliquots of
the same which were previously sonicated for different times (the sonicated component or S
(tS)). Different NS:S (tS) mixing ratios (7:3, 1:1, and 3:7) and sonication times (tS=5, 15, 30
min) were explored. Small-deformation oscillatory measurements (frequency sweeps between
0.01 and 12 Hz) were performed with an ARG2 rheometer operated at 25 C (TA
Instruments, cone geometry: 2 angle, 40 mm diameter, and 54 m gap). A 10% shear strain
was applied in order to ensure working in the linear viscoelastic region (which was
determined in preliminary experiments). Each individual test was completed in about 26 min.
The general flow behavior of the samples was further studied through rotational tests.
To this end, steady-shear experiments were performed at intermediate shear rates (𝛾̇ ≈0.1-300
s-1). Logarithmic ramps (which reduce the initial acceleration and instrument inertia) were
applied to increase/decrease the applied torque. In all cases, the samples were covered with a
thin layer of paraffin oil to prevent from evaporation. Every test was completed in about 1
hour (30 min to vary the applied torque in each sense). Each rheological measurement was
performed in triplicate.
5.2.4 Atomic force microscopy (AFM)
The morphology of the colloidal mixtures was investigated at the solid-gas interface
with a PicoLe 5100 atomic force microscope (Agilent Technologies USA) operated in tapping
mode. PPP-NCH-20 n+-silicon cantilevers from Nanosensors (Switzerland) with the following
characteristics were used: high aspect ratio with a tip height of 10-15 μm and radius of
curvature <10 nm, low spring constant (10-130 N/m), and high resonance frequency (204-497
kHz). The mixtures were diluted 103 times and 100 μL aliquots were subsequently dropcasted onto freshly cleaved discs of mica (Muscovite V-4, 15 mm diameter). The samples
were air dried for about 20 min, then, washed with ultrapure water and dried under a nitrogen
stream. The resulting substrates were scanned in topography and amplitude with a resolution
64
of 512 x 512 pixels over representative regions of 5 x 5 μm2. The raw AFM data was
processed using the software Gwyddion 2.25. In order to correct bow/tilt artifacts, the images
were flattened line-by-line in the horizontal direction with a quadratic polynomial. Caribbean
and green color palettes were added to the topographic and amplitude images, respectively.
Gwyddion was also used to extract relevant statistical parameters such as the average rootmean-square surface roughness (RMS) and the average height (HAV).
5.3
RESULTS AND DISCUSSION
5.3.1 DLS Studies
The colloidal particle distributions calculated for the sonicated samples are displayed
in Figure 1a. For simplicity, only the curves for tS=4 (red solid line), 8 (green), and 20 min
(blue), are shown. The distribution for the non-sonicated sample (tS= 0; i.e. NS-CHIT90) has
also been included for comparative purposes (black solid line). For each single peak, the
centre values of the Rh and the amplitude of scattered light have been extracted (Table 1).
Starting with the analysis of the NS-CHIT90 sample, three major bands centered at Rh = 0.02
(peak 1), 0.18 (peak 2), and 19.5 μm (peak 3) can be distinguished. This evidence indicates
that this commercial product is characterized by a high degree of polydispersity what: (1) is
well supported by the high polydispersity index derived from the mathematical analysis of the
data (PdI=3), and (2) agrees with the conclusions of a previous investigation on the
morphology of this material (SOUZA, H. K. S. et al., 2013). The atomic force microscopy
(AFM) data published in the latter work unveiled the presence of low-M chitosan, under the
form of spherical nanoparticles of about 300 nm in diameter (LMCH-NPs), lying on top of a
polymeric matrix with dimensions in the microscale (HMCH-microfibers). The scanning
electron microscope (SEM), confirmed the coexistence of HMCH-microfibers and different
types of LMCH, mainly: (1) nanofibers of about 150 nm, and (2) much smaller spherical NPs
of 10-20 nm (not seen in AFM). Hence, the size distribution derived from the DLS
measurements in this study; which supports the coexistence of HMCH-microfibers (peak 3)
and LMCH-NPs of medium (peak 2) and small size (peak 1); is in good agreement with the
microscopic evidence reported there.
65
Further analysis of the DLS data also returned the percentages of light intensity
scattered by each individual type of colloid (Figure 1b and Table 1). The results show that up
to a 48% of the intensity scattered by NS-CHIT90, occurred through the HMCH-microfibers
(open squares in Fig 1b). In parallel, the LMCH-NPs of medium size were responsible for a
similar percentage (46%, open circles). In big contrast, the smallest NPs contributed only to a
5.7% of the scattered intensity (explaining, perhaps, why they were not seen under the AFM
in the referred work). In conclusion, the commercial chitosan used in this study can be
roughly considered as composed of HMCH-microfibers and LMCH-NPs (of about 200 nm) in
a 1:1 ratio. The effect of ultrasonication on this colloidal system can be inferred, in first place,
from the evolution in the intensity of the peaks in Fig 1a. By increasing tS, the intensity of the
light scattered by the HMCH-microfibers was dramatically reduced as indicated by the
decrease of peak 3. In contrast, the scattering induced by both types of LMCH-NPs was
shown to increase (peaks 1 and 2). Nonetheless, while the contribution of the smallest NPs
did it in a very slightly way (as indicated by the small changes in peak 1), the intensity
scattered by the medium-sized NPs increased significantly. These observations support the
continuous degradation of the HMCH-microfibers in favor of a concomitant increase in the
population of LMCH-NPs (especially those of medium size).
This fact is not only accounted by the discussed increase of the light scattered by
LMCH-NPs with peak Rh (i.e. through the increase of peak intensities) but may also be
reflected in an increase of light scattered by NPs with Rh in the vicinity of the peak values (i.e.
through peak broadening). Such a behavior was noticed after increasing tS from 4 to 8 min. In
contrast to the expected behavior, the intensity of peak 2 decreased. However, it was
significantly widened as well (the integrated area grew from 7.3 to 10.4 nm, see Table 1).
This evidence suggests that, for this specific experiment (8 min), sonication produced slightly
more polydisperse patterns characterized by larger contents in colloids with Rh surrounding
the central peak values (further investigation is still required but experimental issues such as
the lack of a full control of the temperature in these experiments, could underlie this
behavior). After all, the relevant fact is that the integrated areas shown in Table 1 for both
peaks confirm the increased populations of LMCH-NPs compared to the S (4 min) sample,
thus, following the general trend described above.
Obviously, the scission of the HMCH-microfibers to LMCH-NPs can also induce
slight shifts in the positions of the peaks as observed in the figure. The average peak values
calculated in the range 0-30 min are: 28 ± 10 (peak 1), 220 ± 50 (peak 2), and 18000 ± 3000
66
nm (peak 3). The evolution in the contribution of each type of colloid to the scattering of light
was studied as function of tS. Figure 1b shows the percentage of intensity scattered by
HMCH-microfibers (open squares) and LMCH-NPs of medium (open circles) and small size
(open triangles) in the samples irradiated for different times. Despite the complexity of the
samples, correlation factors as high as R2>0.97 were obtained from the linear regression of the
data registered for the two major colloids (i.e. the microfibers and the medium-sized NPs).
According to these results, the contribution of the microfibers to the scattering of light
drops linearly when tS is increased. Concomitantly, a linear increase in the % intensity of
medium-sized NPs is also noticed. It is noteworthy that the slopes were nearly identical in
both cases (in absolute value, 1.57 vs 1.64 min-1, respectively). This evidence seems to
confirm that the degradation of HMCH by sonication feeds the increase in the populations of
the LMCH-NPs. As it was suggested from the little changes detected in peak 1 (Fig 1a), the
modest increase in the % intensity of the smallest NPs seems to confirm that the degradation
of HMCH-microfibers in NS CHIT90 mostly yields LMCH-NPs of medium size. The general
conclusions are: (1) sonication degrades HMCH-microfibers in CHIT90 at an apparent
constant rate, (2) degradation seems complete for tS<25 min (beyond this value, % intensity is
almost negligible), (3) the degradation of the HMCH-microfibers mainly produces, at nearly
identical rate, LMCH- NPs with average size of 220 ± 50 nm, (4) the smallest NPs (28 ± 10
nm) appear to be a minor product of sonication under the studied conditions.
5.3.2 Mechanical Tests under Dynamic Shear
Mechanical spectra were recorded for the mixtures (colored symbols in Figure 2)
prepared with 7:3 (up triangles), 1:1 (down triangles), and 3:7 ratio (stars) by sweeping the
angular frequency of the shear (ω). The S components were previously sonicated for tS=5
(panel a), 15 (panel b), and 30 min (panel c). The storage (G´) and loss (G´´) moduli are
represented in Figure 2 by filled and open symbols, respectively. The measurements
performed for the individual NS and S (tS) components (black squares and circles,
respectively) have been also included in each panel for comparison purposes. As seen in the
figure, G´´ exhibited higher values than G´ in every case. Only in the case of the NS sample,
both curves showed to converge at high ω (with a hypothetical crossover beyond the upper
67
limit of ω =100 rad s-1). According to Steffe et al. (1996) (HANAOR et al., 2012), this
behavior is typical of viscoelastic fluids. In contrast, as stated by Rao et al. (2010) (TANG, H.
et al., 2010), the energy required for deforming liquids with G´´ >> G´ is dissipated viscously
what induces a liquid-like behavior. The data collected for the isolated S (tS) components
illustrates well how by increasing tS: (1) the magnitude of both moduli decreased in the whole
frequency range, and (2) the (log G´´- log G´) differential followed a clear increase.
These effects were already unveiled in our previous work and were attributed to a
continuous decrease in the viscoelasticity of the solutions occurring in parallel to the increase
in the irradiation time (SOUZA, H. K. S. et al., 2013). This behavior indicated a continuous
decline of the entanglement as the degradation progresses. As the sonication of chitosan does
not change significantly the DD, then, the reduction in the entanglement of the biopolymer
has to be primarily ascribed to the decrease in the average molecular mass and the
corresponding effects in the size and morphology of the colloids (which were investigated
above with the DLS technique). It is noteworthy that the continuous decrease of the molecular
mass under sonication was demonstrated in the referred work from viscosimetric
measurements (SOUZA, H. K. S. et al., 2013). These conclusions become more evident after
analysis of the tan  vs. ω curves in Figure 3. This parameter, which was calculated from the
data in Fig 2 as tan  = G´´/G´, reflects the overall elasticity of the samples and is expected to
decrease as the elastic modulus of the studied material increases. As shown in Figure 3a for
the isolated components (filled symbols), the lowest values were registered for the NSCHIT90 (black squares) what confirms the greater elasticity of this sample. By increasing tS
from 0 (black squares) to 30 min (blue diamonds), tan  increased progressively reflecting the
reduction in elasticity (increased liquid-like behavior) of the sonicated samples.
The data calculated for different mixtures are presented in Figure 3b (colored open
symbols). Regardless of the applied tS, reducing the amount of NS-CHIT90 in the mixtures
resulted in a progressive decrease of the elasticity (increased values of tan  ) in the whole
frequency range. The poorest viscoelastic properties were always found for the 3:7 mixtures
(open stars) which contain the larger fraction of sonicated components. Moreover, the lowest
moduli and the highest tan  values (compared to the pure NS), were found for the mixtures
prepared with S (30) (blue open symbols). As discussed in section 3.1, this component seems
to be the richest in LMCH-NPs and the poorest in HMCH-microparticles. Hence, depending
on the applied tS and the NS:S (tS) ratio, mixtures can be obtained exhibiting different
68
viscoelastic properties (as clearly inferred from Figs 2 and 3) but always within the limits
defined by the response of their individual components. Indeed, from all the investigated
mixtures, only those prepared with S (15) and S (30) in 7:3 ratio, exhibited a similar
mechanical response. All this is indicating that, through the proper selection of these
parameters, the response of the colloidal mixtures to a dynamic shear can be fine-tuned to a
desired degree not achievable by their single isolated components.
In this sense, whereas the mixtures prepared with S (5) showed to behave more closely
to the NS-CHIT90 (see Figs 2a and 3b), the ones prepared with S (15) and S (30) presented
viscoelastic properties closer to the ones of their pure S components (Figs 2b, 2c, 3a and 3b).
In summary, the viscoelastic properties of the commercial chitosan investigated in this work
can be tweaked via its mixing with the products of its own sonication. In this respect, the
elasticity of the resulting mixtures can be effectively reduced by: (A) increasing the fraction
of the sonicated components, and/or (B) increasing the time of exposure of CHIT90 to the
ultrasounds.
5.3.3 Steady Shear Measurements
Further rheological characterization was performed through the implementation of steadyshear methods. Flow curves (apparent viscosity, ηA, vs shear rate, 𝛾̇ ) were recorded by using a
steady state flow ramp in the 0.1-300 s-1 range of shear rate (Figure 4). From the perfect
match found in all cases for the increasing (filled symbols) and decreasing (open symbols)
flow curves, it is evidenced a time independent flow behavior which is typical of nonthixotropic fluids. In line with the behavior expected from the dynamic data presented in the
previous section, the highest values of ηA in the whole range of shear rate were exhibited by
the NS component (black squares in all panels). In the case of the S (tS) components, ηA
showed to decrease (from the high levels of NS) with increasing tS (black circles in all panels).
The curves registered for the NS and S (5) samples exhibited two distinct regions (black
squares and circles in Fig 4a): (A) in the low 𝛾̇ range, ηA is nearly shear-independent what
indicates a Newtonian-like behavior; and (B) for higher 𝛾̇ , ηA decreases following a power
law which evidences a typical shear-thinning flow behavior.
69
In the first zone, the disruption of intermolecular entanglements by the action of shear
force is well balanced by the formation of new ones so that no net change occurs and the zeroshear viscosity is maintained. However, by increasing 𝛾̇ , entanglement disruption turns
predominant over formation and the molecules tend to align in the direction of flow what
results in a decrease of the viscosity. Increasing tS, not only resulted in a shift of this transition
to higher values of 𝛾̇ but also reduced progressively the contribution of the shear-thinning.
This is well illustrated by the almost pure Newtonian response registered for the S (30)
sample (black circles in Fig 4c). Agreeing with previous interpretations, this behavior must be
due to a significant decrease in the size of the chitosan colloids (CHUNG et al., 2004;
COTTER et al., 2005). Regarding the behavior of the mixtures, the viscosity clearly dropped
with decreasing the mixing ratio whatever was the S (tS) component studied. In fact, the
lowest viscosities were consistently found for the 3:7 mixtures.
Whereas the magnitude of all these changes was relatively small in the mixtures
containing S (5) (which exhibited ηA values very close to those of the NS CHIT90 at any
mixing ratio), much larger variations occurred in the mixtures of S (15) and S (30). In fact,
agreeing with that found in the dynamic tests, the lowest viscosities were consistently found
for the richest mixtures in these components (see Figs 4b and 4c). Shear-thinning was also
shown to decrease in these mixtures by decreasing the mixing ratio. In particular, while the
mixtures of S (5) and S (15) still exhibited some sort of power law decay, the richest mixture
in S (30) showed an almost negligible shear-thinning. In general, these results are fully
compatible with the conclusions drawn in the previous section. Polymeric fluids undergoing
transitions from Newtonian to shear-thinning behavior are usually modeled with the Cross
and Carreau equations, respectively (BASTOS et al., 2010; CARREAU, 1972; SOUSA et
al., 2013; TOMASIK; ZARANYIKA, 1995):
ηA= η + [(η0-η)/1+(τ∙𝛾̇ )M]
(1)
ηA= η + [(η0-η)/(1+(λ∙𝛾̇ )2)N]
(2)
where ηA, η0, and η∞, are the apparent, the zero-shear rate, and the infinite-shear rate
viscosities, respectively, in units of Pa∙s; τ and λ are time constants given in s; and M and N
are dimensionless constants related to the power law exponent n (M=1−n and N=(1−n)/2, with
0≤N<0.5 for η∞<<ηA<<η0). Assuming that η0>>η∞ (in this study ηA was never close to η∞),
equations 1 and 2 can be simplified to:
70
ηA= η + [η0/A]
(3)
with A=1+(τ∙𝛾̇ )M (Cross) or A=(1+(λ∙𝛾̇ )2)N (Carreau).
The data collected during the increasing shear rate excursion (filled symbols in Fig 4)
were fitted to the simplified Cross (grey straight lines) and Carreau (violet dashed curves)
models. Table 2 summarizes the fitting data and includes the residual sum of squares (RSS)
and the mean relative deviation (MRD) to assess the quality of the correlations. In general, as
it is indicated by the low values obtained for these parameters, the steady shear data gathered
for the isolated components and their mixtures fitted reasonably well to both models. The
slightly lower values returned from the fittings to the Cross model indicate a slightly better
suitability of the latter to describe the studied systems. Newtonian fluids are typically
characterized by negligible values of M and . Therefore, the non-negligible values derived
from these fittings must be taken as an indicator of the degree of departure from a purely
Newtonian behavior. In this regard, the decreasing relevance of shear-thinning upon
increasing tS matches perfectly with the concomitant decrease of these constants in the S (tS)
series. In the case of the mixtures, and irrespective of the applied tS, the constants decreased
in parallel to the increase in the fraction of the sonicated components.
On the other hand,  and  can be regarded as relaxation constants that indicate the
onset for shear-thinning. Their drastic reduction in the sonicated components (in respect to the
maximum values recorded for NS sample), confirms the great effect of the ultrasonic
treatment on the viscoelastic response of the colloidal suspensions of the biopolymer. In the
case of the mixtures, the magnitude of the returned constants fell always within the range of
values delimited by those for their NS and S components. Nonetheless, while the relaxation
times for S (5) mixtures were only slightly lower than those found for NS, in the case of S(15)
and S (30), the reduction fell closer to the values registered for the pure sonicated
components. Interestingly, these parameters exhibited very little dependence on the mixing
ratio.
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5.3.4 Cox-Merz Rule and Temperature Effect
Despite there is no theoretical link between the data coming from the mechanical tests
under steady and dynamic shear conditions, Cox and Merz found a very good empirical
correlation between them in certain systems (COX and MERZ,1958). If the rule applies, any
of both datasets can be predicted from each other. More specifically, a good correlation was
found between the magnitudes of the apparent viscosity in steady shear () and the complex
viscosity in oscillatory shear (*), compared at equal values of shear rate and frequency,
for solutions of random-coil polysaccharides and of some synthetic polymers (TANG, H. et
al., 2010). Nevertheless, even in these cases, the rule is not followed at higher frequencies
where significant departures between both parameters are usually found. Deeper exploration
unveiled that the rule generally fails in systems like Boger fluids, dilute solutions, crosslinked or gelled systems, and particulate/aggregated dispersions (RICHARDSON; ROSSMURPHY, 1987). In order to study its applicability to the investigated systems the apparent
(A), complex (*), and dynamic (’) viscosities for samples NS, S (tS), and their 1:1
mixtures, are plotted in Figure 5. The data gathered for samples NS, S (5), and its 1:1 mixture
(red data), illustrate how A departed significantly from both ’ and *in the whole ω / 𝛾̇
range.
The deviation of A has been attributed to structure decay due to the effect of the strain
deformation applied to the system or to different types of molecular rearrangement occurring
in the flow patterns over the applied shear (RICHARDSON; ROSS-MURPHY, 1987). In the
latter case, the behavior may be interpreted in terms of specific interpolymer chain
interactions occurring in addition to entanglements. These results indicate that, according to
the evidence discussed above, the Cox-Merz rule may be not followed by those mixtures
characterized by a high elasticity and a high content in HMCH-microfibers (and their
aggregates). Hence, these results would agree with the failure of the rule found in other
particulate/aggregated systems and, more specifically, in the case of biopolymer dispersions
with hyper-entanglement (i.e. high density of entanglements) (CHAMBERLAIN; RAO,
1999). Interestingly, the differences between the viscosities under steady and oscillatory shear
were clearly mitigated when tS was increased. Accordingly, a good correlation was found for
S (15) and S (30) and their corresponding mixtures within the range 0.1-10 s-1.
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Some discrepancies can be found in the literature regarding the applicability of the
rule in chitosan-based systems. For instance, Calero et al reported good correlation in chitosan
solutions at a temperature of T=25 ºC but a rule failure whenever it was decreased to T<20 ºC
(TOMASIK; ZARANYIKA, 1995). On the other hand, Cho et al found the rule´s
applicability to depend upon the changes in the ionic strength (I) of the system (CHO, J. et al.,
2006). These results are compatible with the behavior shown in Fig 5: i.e. chitosan
solutions/dispersions with strong Newtonian character seem to follow the Cox-Merz rule
better than those which exhibit enhanced viscoelastic properties. Although in our case these
differences seem mainly determined by the changes in M induced by sonication, there is no
contradiction with literature data in the sense that other experimental conditions (T, I, etc) and
chitosan properties (concentration, DD, etc) can also influence the flow behavior of aqueous
polysaccharide solutions/dispersions and, thus, the applicability of the rule.
5.3.5 AFM Studies
The atomic force microscope was used to investigate the morphological characteristics
of NS:S (tS) mixtures. For this purpose, freshly cleaved mica substrates were modified with a
thin colloidal film of each mixture by drop casting. Figure 6 shows the topographic (panel A)
and the equivalent amplitude images (panel B) obtained for the prepared films. A quick
glance at panel A reveals clear differences in the topography of the different samples.
Although a more detailed discussion is given below, these variations appear to be correlated
with the changes made in the mixing ratio and sonication time. Starting with the mixtures of S
(5), the topography of the 7:3 mixture (Fig 6AA) does not look anything like the typical
image of a bare mica surface (see supplementary information). The presence of a densely
packed film of chitosan (which, indeed, looks like scraped at different locations) is strongly
suggested. Its thickness has been estimated in 2 nm from the extracted height profiles. Above
this layer, non-branched fiber-like structures with length ranging between 0.5 and more than 1
μm, height around 4 nm, and width about 40 nm, can be distinguished. Apparently, these can
form thicker bundles of about 100 nm which organize in local networks (as can be
distinguished in the top-left part of the image).
73
The high packing density of the background layer and the observation of individual
fibers on top, are consistent with the AFM and SEM evidence reported for pure films of NS
CHIT90 and other biopolymers, e.g. agars (SOUZA, H. K. S. et al., 2013), when prepared
from relative dilute solutions (<30 μg·mL-1). Two other types of features can be found in the
outermost layer: (1) small particles with diameter and height of about 50 and 6 nm,
respectively (tagged as #1 in Fig 6AA), and (2) larger odd-shaped bodies with size larger than
150 nm and height about 10 nm (tagged as #2). Based only on the topographic evidence, it is
hard to ascertain whether these particles correspond to LMCH-NPs, fibril aggregates, or
protuberances from the underneath layer. It is worth remembering that the DLS data in section
3.1 unveiled the presence of a high % of LMCH-NPs of about 200 nm in the NS CHIT90
sample and the consistent increase in its population under sonication. Hence, their observation
under the AFM would make sense. Comparing the height of the individual fibers and the
underlying film, it is hypothesized that the latter may consist of a monolayer of selfassembled HMCH-microfibers or microfiber-NP conjugates (this mixture contains up to 70%
of the fibrous NS CHIT90 component).
The analysis of the amplitude scan (Fig 6BA) sheds more light into some of these
issues. For instance, the individual fibers appeared much better defined in terms of shape,
thus, dispelling any doubt about their morphology. Nevertheless, in great contrast with the
topographic evidence, a relatively flat and unscratched underlayer is shown. This is in line
with the relatively low surface roughness derived from the statistical analysis of Fig 6AA
(RMS=0.50 nm, see other statistical parameters in Table 3). Hence, the crack-like features
found in that image should be alternatively interpreted as edge-overshoot artifacts in the z
axis; i.e. an exaggeration of the edges along stepped profiles as a consequence of the piezo
hysteresis (one of the most common issues in AFM). As for the origin of the spherical
features, although the evidence in Fig 6BA is still not conclusive, actually, it seems to support
the hypotheses of the aggregated and/or protruding fibrils. Nonetheless, even if these features
were undoubtedly attributed to LMCH-NPs (originally present in the NS CHIT90 or coming
from its ultrasonic fragmentation), the truly relevant fact is that their relevance in the colloidal
films prepared from this mixture is relatively residual.
As it can be seen in the images, significant changes were triggered by increasing the amount
of S (5) in the mixture (panels B and C in Figs 6A and 6B). In fact, two clear trends are
derived from the simple eye inspection of the images:
74
(1) While the amount of fibers falls sharply (only one of these features is found in Figs 6AB
and 6BB), the presence of spherical NPs increases notably in the outermost layer.
(2) The packing density at the underlayer follows a significant decrease.
In spite of the increasingly nanoparticulate texture exhibited by the outermost layer,
especially in the case of the 3:7 mixture (Figs 6AC and 6BC), the microfibers are still
distinguished as part of the underlayer. In fact, in this specific mixture, the fibers can be
traced by following the NPs deposited in the upper layer along their 1D length (as better
clarified in the amplitude image). The described transitions are well supported by the
statistical data (Table 3). Accordingly, the overall change in the surface texture is perfectly
matched by the increase of RMS and HAV , which reached values of 0.79 and 3.05 nm in the
case of the 3:7 mixture. It could be argued whether the data for the 1:1 system (which showed
a slight decrease in RMS, and a barely unchanged HAV) are against this trend. Nevertheless, it
has to be pointed out that the presence of NPs on surface was only predominant for the 3:7
mixture. In fact, compared to the 7:3, the images of the 1:1 film do not only show a negligible
amount of fibers but also a similar amount of NPs, which explains the small changes in RMS
and HAV. From this analysis, it is postulated that the increase in the fraction of LMCH-NPs
(with respect to the HMCH microfibers) induced upon the increase in the mixing ratio is
responsible for the changes observed in the morphology. This would be backed by the DLS
data presented above which demonstrated that the sonicated samples contain an increased
population of NPs coming from the fragmentation of the microfibers.
Hence, the increasing fraction of low-M forms of the biopolymer in the mixtures not
only explains the lower amount of fibers imaged on surface (and, conversely, the higher
amount of NPs) but also the decrease in the packing density of the underneath layer. This
seems determined by two main factors: (a) the lower availability of fibers in the mixtures for
self-assembly, and (b) the intercalation of the fibers, and/or their bundles, by NPs (an example
is indicated by the red arrows in Figs 6AC and 6BC). The role of tS on the morphological
aspect of the mixtures can be derived by comparison of the images in the central and right
columns (15 and 30 min) with those placed at their left (5 min). But before proceeding with
the statistical analysis of the images, let us emphasize that increasing tS over 5 min resulted in
a virtual demise of the microfibers from the outermost layer (although they seem to be still
part of the underlayer). Regarding the effect of tS in the NS-rich samples, i.e. the 7:3 mixtures,
there was a clear transition from a landscape dominated by the presence of fiber-like features
75
at tS=5 min, to different scenarios for tS= 15 (Figs 6AD and 6BD) and 30 min (Figs 6AG and
6BG) characterized by an increasing predominance of spherical NPs.
In great contrast to that expected from the DLS results and the previous research on
sonicated CHIT90 (SOUZA, H. K. S. et al., 2013), the size of the largest particles increased
from 130 nm in image D to about 250 nm in E (as measured from the amplitude image) and
the height (extracted from the leveled topographic images) raised from 12 to 40 nm. Such an
increase can only be interpreted in terms of an enhancement of aggregation phenomena in the
drop casted films. These processes can occur in solution (prior to casting) or through a
dewetting-driven effect which involves the formation of microdroplets (KRISHNAN, 2001;
SOUZA, H. K. S. et al., 2013). Any of these processes would be likely boosted upon an
increase in the fraction of LMCH-NPs as a consequence of prolonged sonication. Hence,
because of the reinforcement of colloidal aggregation phenomena, the AFM images would
exhibit increasingly larger particles just as verified in Fig 6. This hypothesis also explains the
lower amount of small NPs (20-40 nm) found in the images of S (30) compared to S (15)
(images G and D in Figs 6A and 6B). The analysis of images E-H and F-I can provide us with
a good perspective on the effects of increasing tS in the mixtures with larger fractions of the S
components. To this end, amplitude images are more appropriate as their topographic
counterparts are slightly affected by horizontal artifacts from the polynomial leveling. Starting
with the 1:1 system, it was observed that the size and height of the largest particles observed
in the image for S (30) (panel H), which fell at about 150 and 12 nm, respectively; were
smaller than those in image G. Comparison with image E is complicated because, in this case,
a highly homogeneous composite (where the individual fibers and NPs are difficult to
distinguish from each other) was apparently formed.
In the case of the 3:7 mixtures, no apparent differences in size and height were inferred
for the NPs displayed in images F and I (which fall, in both cases, within the range of 20-40
and 1-2 nm, respectively). Furthermore, these are much smaller than the largest particles
found in G and H. These results suggest that the effect of tS in the enhancement of aggregation
processes was considerably reduced for the 1:1 mixture and almost completely lost for the 3:7
system. The correlation between the relevance of aggregation phenomena observed for the
different mixtures of S (30) and their content in NS CHIT90, indicates a potential involvement
of the HMCH-microfibers in the formation of aggregates. Hence, the large particles must be
ascribed not only to LMCH-NPs self-aggregation but also to a possible formation of HMCHLMCH conjugates. In summary, a morphological transition, from a starting material which
76
seems quite rich in microscopic fibers to others with an aspect increasingly more
nanoparticulated, is triggered by increasing tS or through the variation in the NS:S (tS) mixing
ratio. The variety of intermediate morphologies identified in these experiments turns these
systems into very promising precursors for the fabrication of chitosan biofilms with tunable
properties. Within this context, the 1:1 mixture prepared with S (15) (central image in both
panels) exhibited a relatively homogenous structure.
5.4.
CONCLUSIONS
This work proposes the preparation of blends of colloidal chitosans with different
morphology and molecular mass (M), as alternative precursors for the fabrication of food
packaging films with fine-adjusted properties. Specifically, a commercial chitosan
characterized by a high average molecular mass (M≈300 kDa) and DD=90% was mixed in
aqueous solution with the products of its own molecular fragmentation. In this respect, the
ultrasounds technique has played a central role in our strategy as an affordable tool to execute
the degradation of the biopolymer, and the simultaneous shaping of the resulting colloids,
under controlled conditions. The results herein presented demonstrate that through the control
of the sonication time and the mixing ratio between sonicated and non-sonicated components,
it is possible to adjust the viscoelasticity and the morphological aspect of the mixtures at a
variety of levels intermediate between the limits defined by: (a) the shear-thinning flow
behavior previously reported for non-sonicated CHIT90 (with a characteristic structure rich in
microscopic fibers); and (b) the almost pure Newtonian response of the highly sonicated
samples.
Although further experimentation and testing of the corresponding biofilms is required
to confirm these claims, the research presented in this paper strongly suggests the validity of
the proposed concept. Through the appropriate selection of ts and mixing ratio, the colloidal
mixtures can be prepared with intermediate properties so that a better balance between the
mechanical properties and the moisture sensitivity could be met in their corresponding films.
Hence, their use as precursors for the fabrication of packaging films to work in direct contact
with foodstuffs may be more advantageous. In this respect, the 1:1 mixture prepared with a
chitosan sonicated for 15 min emerges from this study as the most promising system to be
77
tested in future work (due to its significant viscoelasticity and homogeneous morphology). In
a more general vein, these results evoke the fundamental role of smart design and processing
for the realization of a new generation of additive-free sustainable and fully functional
bioplastics.
Acknowledgements
This work was supported by the European Union (FEDER) and National funds from
the National Strategic Reference Framework Portugal 2007-2013 (QREN) allocated through
the Operational Competitiveness Programme (COMPETE) and the North Portugal Regional
Operational
Programme
(ON.2)
(FCOMP-01-0124-FEDER-37285,
FCOMP-01-0124-
FEDER-041438, NORTE-07-0124-FEDER-000069) to REQUIMTE and CIQ through the
Portuguese
Foundation
for
Science
and
Technology
(FCT)
(projects:
Pest-
C/EQB/LA0006/2013, Pest-C/QUI/UI0081/2013, and EXPL/QEQ-QFI/0368/2013). J.M.C.P.
is also grateful to the FCT for granting him with a post-doctoral fellowship
(SFRH/BPD/75259/2010). L.P.G. acknowledges CAPES (Programa Institucional de Bolsas
de Doutorado Sanduíche no Exterior, PDSE; Proc nº 18935/12-5), FAPERJ, and CNPq for the
concession of a PhD fellowship.
Author Contributions
C.T.A., V.M.F.P., and M.P.G, conceived the project. L.P.G., J.M.C., and H.K.S.S. designed
and performed the rheological, DLS, and AFM experiments. C.T.A., V.M.F.P., M.P.G.,
H.K.S.S., J.M.C., and A.F.S., analyzed and interpreted the corresponding results. H.K.S.S.
and J.M.C. wrote the paper.
Corresponding Authors
Hiléia K. S. Souza (PhD), Tel.: + 351 225 081 884, E-mail: [email protected]; José M.
Campiña (PhD), Tel.: +351 220 402 643, E-mail: [email protected]
78
Graphical Abstract (for review)
79
Highlights (for review)
Highlights
The fragmentation of a fibrous chitosan by ultrasonication was followed with DLS
Colloidal mixtures of the sonicated samples and the original biopolymer were prepared
The viscoelasticity and structure of the mixtures were studied by rheology and AFM
Properties can be tuned through the selection of mixing ratio and sonication time
Excellent perspectives as precursors of fully biodegradable films for food packaging
80
Scheme 1. Artwork depicting the main concepts explored in this investigation. The graphical
representation of the morphologies characterizing the CHIT90 samples, non-sonicated (blue
frame) and after sonication for 60 min (green frame), as well as the properties of their
precursor solutions and corresponding transparent films, have been summarized from the
results reported in Ref. (SOUZA, H. K. S. et al., 2013). A hypothetical morphology (red
frame) and the main questions to answer about the properties of their mixtures are also
presented.
Figure 1. a) The distributions of the amplitude of the scattered light as a function of the
particle hydrodynamic diameter for CHIT90 irradiated with ultrasounds for: 0 (non-sonicated,
black solid line), 4 (red), 8 (green), and 20 min (blue). b) Evolution of the intensity of light
scattered at each of the peaks displayed in (a) upon the gradual increase of the sonication time
(tS). The correlation factors obtained from the linear fittings of the data (black solid lines) are
also included. The 3% (w/w) CHIT90 dispersions were diluted 103 times in all cases before
measurements.
Figure 2. Mechanical spectra gathered for the pure Chit90 solutions of NS (black squares, all
panels) and S (black circles, all panels) sonicated for tS=5 (panel A), 15 (panel B), and 30 min
(panel C). The mixtures of these components are represented by the different colored
symbols: tS=5 (red symbols in panel A), 15 (green symbols in panel B), and 30 min (blue
symbols in panel C). For each system the following NS/S ratios were studied: 7:3 (up
triangles), 1:1 (down triangles), and 3:7 (stars); are also presented. The storage (G’) and loss
(G’’) moduli are represented by the filled and open symbols, respectively.
Figure 3. (a) Evolution of tan as a function of the oscillation frequency (ω) for the isolated
samples of NS (black filled squares) and S treated with tS=5 (red filled circles), 15 (green
filled up triangles), and 30 min (blue filled diamonds). (b) Analogous data obtained for the
mixtures prepared with the following NS/S ratios: 7:3 (open squares), 1:1 (open hexagons),
and 3:7 (open stars). Despite all the mixtures prepared from a given S component are colored
in accordance to (a), arrows and legends have been added for better clarity. tan was
calculated as G´´/G´ from the data in Figure 2.
Figure 4. Steady shear curves obtained for the pure NS (black squares) and S (tS) components
(black circles) treated with tS= 5 (a), 15 (b), and 30 min (c). Filled symbols represent the
increasing shear rate excursion and their open counterparts the decreasing one. The grey
straight line and the violet dashed curve represent the fitting of the filled data to the Cross and
Carreau models, respectively. The behavior registered for the mixtures of these components
are represented by the different colored symbols: tS=5 (red symbols, panel a), 15 (green
81
symbols, panel b), and 30 min (blue symbols, panel c). For each system the following NS:S
(tS) mixture ratios were studied: 7:3 (up triangles), 1:1 (down triangles), and 3:7 (stars)
Figure 5. Comparison between A (solid lines, left scale), *(dashed lines, right scale),
and ’ (dash dot dotted lines, right scale) measured at 25ºC for NS and S (tS) chitosans
prepared with tS= 5, 15, and 30 min (black curves) and their corresponding 1:1 mixtures (red,
green, and blue curves, respectively).
Figure 6. AFM images taken in tapping mode for freshly cleaved mica substrates decorated
with thin films of the different NS:S (tS) colloidal mixtures. The mica discs (drop-casted with
the different colloidal suspensions) were simultaneously scanned in topography (A) and
amplitude (B) over regions of 5 x 5 μm2. The red scale bars are 1 μm.
82
Figure 1
83
Figure 2
84
85
Figure 3
86
Figure 4
87
88
Figure 5
89
Figure 6
90
Table 1. Peak data, hydrodynamic radius (Rh) and Amplitude, derived from the curves in
Figure 1a. Other peak parameters as the percentage of intensity scattered at each peak
(Intensity %) and the integrated peak areas (Area) were calculated with i-Size 2.0 and Origin
7.0 software, respectively. The errors for the peak amplitudes were calculated as the standard
deviation of the consecutive measurements taken for each sample.
Rh / μm
Amplitude
Intensity %
Area / nm
Peak 3
19.5
0.069 ± 0.005
48.0
437.5
Peak 2
0.18
0.035 ± 0.004
46.4
4.746
Peak 1
0.02
0.007 ± 0.003
5.67
0.055
Peak 3
15.3
0.032 ± 0.002
35.2
297.8
Peak 2
0.23
0.047 ± 0.008
57.0
7.328
Peak 1
0.02
0.010 ± 0.001
6.25
0.072
Peak 3
17.9
0.042 ± 0.004
0.56
334.9
Peak 2
0.26
0.040 ± 0.007
0.54
10.42
Peak 1
0.02
0.008 ± 0.002
6.57
0.104
Peak 3
19.5
0.026 ± 0.002
8.61
83.78
Peak 2
0.23
0.049 ± 0.003
82.6
10.45
Peak 1
0.02
0.009 ± 0.001
8.61
0.116
NS-CHIT90
S (4 min)
S (8 min)
S (20 min)
91
Table 2 Parameters returned upon fitting the data in Figure 4 to the Cross and Carreau simplified models.
Cross
o
 (s)
M
RSS

RSS
9.60
0.13400
0.60
0.123
8.4
8.60
0.84
0.18
1.0028
20.1
7:3
8.14
0.12970
0.54
0.142
2.7
7.20
0.70
0.17
2.8240
8.4
1:1
8.02
0.12660
0.53
0.537
5.7
7.02
0.67
0.17
3.9040
9.9
3:7
6.81
0.12287
0.48
0.311
4.9
5.62
0.64
0.15
1.7296
8.5
2.71
0.01287
0.58
0.058
2.6
2.56
0.23
0.11
0.3198
7.6
7:3
1.71
0.00780
0.55
0.067
3.9
1.50
0.09
0.12
0.2314
7.5
1:1
1.58
0.01200
0.35
0.211
8.7
1.20
0.08
0.11
0.0800
5.7
3:7
1.01
0.00300
0.41
0.114
8.2
0.86
0.05
0.09
0.1602
9.1
0.75
0.00160
0.52
0.113
10.4
0.67
0.05
0.07
0.0253
5.5
7:3
1.12
0.00400
0.56
0.070
5.8
1.05
0.08
0.08
0.0329
6.2
1:1
0.51
0.00084
0.54
0.057
12.1
0.49
0.06
0.06
0.0449
9.0
3:7
0.52
0.00100
0.45
0.145
17.4
0.39
0.05
0.05
0.1240
15.0
0.25
0.00001
0.38
0.003
4.7
0.24
0.02
0.02
0.0004
2.1
(Pa∙s)
NS
S (5 min)
NS / S (15 min)
Mixing Ratio
S (15 min)
NS / S (30 min)
Mixing Ratio
S (30 min)
MRD x 10-3 o (Pa∙s)
MRD x 10-
 (s)
Chit 90
NS / S (5 min)
Mixing Ratio
Carreau
3
92
Table 3 Surface roughness (RMS) and average size (HAV) extracted from the analysis of the
images in Fig 6A using the freeware Gwyddion 2.25
tS / min
5
15
30
NS:S Mixing Ratio
RMS / nm
HAV / nm
7:3
0.5
1.8
1:1
0.4
1.8
3:7
0.8
3.0
7:3
1.0
18.3
1:1
0.9
5.3
3:7
0.1
1.0
7:3
2.0
6.5
1:1
0.4
18.4
3:7
0.3
1.4
93
Supplemental Materials on:
Tweaking the Mechanical and Structural Properties of
Colloidal Chitosans by Sonication
Laidson P. Gomes,1,2 Hiléia K. S. Souza,1* José M. Campiña,3* Cristina T. Andrade, 4 Vânia
M. Flosi Paschoalin,2 A. F. Silva,3 and Maria P. Gonçalves1
1
REQUIMTE/LAQV, Departamento de Engenharia Química. Faculdade de Engenharia. Universidade do Porto.
Rua Dr. Roberto Frias, 4200-465, Porto, Portugal.
2
LAABBM, Instituto de Química, Universidade Federal do Rio de Janeiro, UFRJ, Av. Athos da Silveira Ramos
149, CT, Bl A, sala 545, Ilha do Fundão, CEP 21941-909, Rio de Janeiro Brazil.
3
Centro de Investigação em Química da Universidade do Porto (CIQ-UP). Departamento de Química e
Bioquímica. Faculdade de Ciências. Rua do Campo Alegre, 687, 4169-007, Porto, Portugal.
4
Programa Ciência de Alimentos, Instituto de Macromoléculas Professora Eloisa Mano, Universidade Federal
do Rio de Janeiro, Centro de Tecnologia, Bloco J 21941-598, Rio de Janeiro, RJ, Brazil
*
Corresponding author information: Hiléia K. S. Souza (PhD), Email: [email protected], Tel.: + 351 225 081 884; José M. Campiña
(PhD), Email: [email protected] , Tel: +351 220 402 643
94
1. Additional Material: AFM of bare Muscovite Mica:
Figure S1. AFM image of a freshly cleaved mica disc (Muscovite V-4, 15 mm diameter) taken in
tapping mode. The topography of the bare surface was scanned over a representative sample area of
5 x 5 μm2.
2. Discussion
Figure S1 shows the image of a representative region of a freshly cleaved mica disc
surface. Agreeing with other images previously reported for this substrate (Z. Shao and D. M.
Czajkowsky, Journal of Microscopy, 2003, 211,1-7; G. B. Webber et al, Faraday
Discussions, 2005, 128, 193-209), the surface seems completely flat and mostly featureless. A
series of ultra-small dots (with a “tail” in the direction of sample scanning) can be
distinguished from the flat background. The tails indicate that this a type of tip-related
artifact. It is well-known that fast scanning hydrophilic surfaces, like muscovite mica, at
regions covered with ultrathin layers of water can result in tip-sticking issues as it appears to
be reflected in the image. After all, the impact of these artifacts on the image was small so
that the general characteristics of the substrates are clearly inferred from the image.
95
6
CAPITULO IV – ESTUDO ORIGINAL 2
FABRICATION AND CHARACTERIZATION OF FIBERS CHITOSAN-BASED
FILMS REINFORCED WITH CHITOSAN NANOPARTICLES
Laidson P. Gomes1,2, Hiléia K. S. Souza*2, José M. Campiña*3, A. Fernando Silva3, Cristina
T. de Andrade4, Maria P. Gonçalves2 and Vânia M. F. Paschoalin1
1- LAABBM, Universidade Federal do Rio de Janeiro – Instituto de Química – Avenida
Athos da Silveira Ramos 149 – 21949-909, Rio de Janeiro, Brazil
2- REQUIMTE, Departamento Engenharia Química, Faculdade de Engenharia, Universidade
do Porto, Rua Dr. Roberto Frias, 4200-465, Porto, Portugal
3- Centro de Investigação em Química (CIQ-L4), Departamento de Química, Faculdade de
Ciências, Universidade do Porto, Rua do Campo Alegre 687, 4169-007, Cidade do Porto,
Portugal
4- Universidade Federal do Rio de Janeiro – Instituto de Macromoléculas Professora Eloisa
Mano, Universidade Federal do Rio de Janeiro, Centro de Tecnologia, Bloco J 21941-598,
Rio de Janeiro, RJ, Brazil.
*Corresponding author:
Abstract
Blend films composed of chitosan-fibers (CHIT90) and sonicated chitosan (S) were
prepared by the knife coating method. The sonicated chitosan were obtained from CHIT90
(NS) (DD = 90%) fragmented by ultrasound. Chitosan nanoparticles obtained in variable
extensions (ts = 5, 15 and 30 minutes) were used as reinforcing agents in chitosan fibers films.
To improve the mechanical properties of biofilms chitosan were reinforced with nanoparticles
The biofilms were produced by a homogeneous series of mixtures between NS/S(ts) at
different ratios (3:7, 1:1 and 7:.3) with the same distribution of degrees of polymerization and
without the incorporation of additives or plasticizers. The influence of nanoparticles contents
on the microstructure, on mechanical and barrier properties, moisture sensitivity, transparency
and color of the blend biofilms were analyzed. The biofilms were investigated by scanning
96
electron microscopy (SEM), which showed a homogeneous morphology. The mechanical
strength and stiffness of transparent biofilms were positively affected when stress tests were
applied. The addition of nanoparticles improved the mechanical properties of films. The
produced biofilms presented superior elongation at break. Sensitivity to moisture was
significantly decreased through water vapor permeability measurements and water sorption
isothermal data with the decreasing of molecular mass from biofilms constituents. These
findings may be useful for the future design of bioplastics with improved properties, as well
as for the development of biocompatible nanoparticles with tunable size and molecular mass.
Addition of nanoparticles improved the mechanical properties of films.
Keywords: Chitosan, Biofilms, Nanoparticles, Mechanical proprieties, Water vapor
permeability, Scanning electronic microscopy.
6.1
INTRODUCTION
There is a worldwide interest in replacing the use of oil-based synthetic plastics with
biodegradable, nontoxic and edible materials in packages. The development of new package
products can benefit various industrial activities, particularly the production, distribution and
commercialization of food (DAVIDOVICH-PINHAS et al., 2014). Chitosan (CH), a
polysaccharide derived from chitin, is a promising biopolymer since chitin is the second most
abundant polysaccharide in nature and can be obtained as a reject of the seafood industry in
coastal regions, inland or even associated to shrimp aquaculture production. Besides the
environmental benefits related to the removal of seafood residues and the replacement of
petroleum-based-packages (CHANTARASATAPORN et al., 2013), chitosan can be
considered an active package material, since its physicochemical properties, such as
molecular weight and degree of deacetylation, can confer special activities to chitosan,
including antimicrobial activity, which can be very useful in food packing (COOKSEY,
2005). The polycationic nature of these polymers also allows the association with natural
antibiotics or antioxidants, reinforcing the applicability of these polymers in food preservation
(FAN et al., 2014). Chitosan biofilms can be used in multi-layer packaging and provide
opportunities for new product development (DEBEAUFORT et al., 1998).
97
In fact, the fabrication of transparent CH bioplastics for food packaging, and other
applications has been widely studied (SOUZA, H. K. S. et al., 2013). Unfortunately, pure CH
biofilms suffer from poorer mechanical properties and enhanced moisture sensitivity than
their oil-derived counterparts (PENG; LI, 2014). The use of certain plasticizers (ethylene
glycol, for example) has solved some of these limitations (SRINIVASA; RAMESH;
THARANATHAN, 2007). However, possible transfer to food poses a health threat. Recently,
irradiation of high molar mass CHs by ultrasonication has been shown to transform fibrous
structures in the range of microns (CFBs) to tiny spherical nanoparticles (CNPs) (LU et al.,
2013b) CHs treated with increasingly longer sonication times yield biofilms with improved
moisture resistance but degraded mechanical properties (ANTONIOU et al., 2015).
With the purpose of reducing moisture sensitivity and still retaining significant
strength and flexibility (with no aid of plasticizers), an approach based on the use of fiber
blends and nanoparticles is presented. The focus of our study was to improve the fiber
mechanical properties without affecting their biocompatibility. In the proposed treatments, we
sought to reduce fiber diameters to produce a compact structure and improve their strength
properties. To this end, CH was submitted to controlled fragmentation to CNPs by
ultrasonication at different time intervals (ts = 5, 10 and 30 min). The resulting solutions were
mixed with unmodified CHIT90 (NS) at different ratios (NS/S(ts): 3:7, 1:1, 7:3) and their
biofilms prepared through the knife-coating method. The physical, functional and barrier
properties of the blend films were correlated with the amount of each component of the
mixture. Mechanical properties were assessed by tensile tests and moisture sensitivity was
evaluated by contact angle measurements of water vapor permeability and water sorption
isothermal measurements. Morphology was also studied by scanning electron microscopy
(SEM). The results evidenced enhanced moisture resistance and improved mechanical
properties compared to biofilms prepared from their individual precursor species. It was
showed that reduction in fiber diameters was due to the formation of a crystalline structure
and was translated into improvement in fiber strength.
6.2
MATERIAL AND METHODS
6.2.1 Material and solutions
Chitosans 150-200 kDa with DD 90% (CHIT90) were purchased from Primex
(Siglufjordur, Iceland). Chitosan 3 % (w/w) were solved in 0.1 M acetate buffer pH 6.0
98
prepared with distilled water and the pH was adjusted by the addition of drops of glacial
acetic acid or sodium acetate. After stirring for 2 h, the solutions were stored at 4 ºC. Before
measurements, Chito90 stock solutions were incubated at room temperature for 60 min and
subsequently centrifuged at 21,000 x g (9 ACC, 25 ºC) for 30 min using a Beckman Coulter
centrifuge (Model Alegra 25R).
6.2.2 Ultrasonic rupture of chitosan fibers
Thirty mL of the Chito90 stock solutions were submitted to ultrasonic irradiation (S)
on ice for defined period of time (ts= 0, 5, 15 and 30 min) using an ultrasonic probe (Sonic,
model 750 watts) equipped with a 1/2” microtip (constant duty cycle and 40% amplitude)
with intervals of 1/1s. Measurements were performed in triplicate.
6.2.3 Mixing solutions
The filming-forming solutions of low molar mass chitosan (LMm chitosannanoparticles) prepared by sonication at different sonication times (S (ts)) were mixed with
the stock solution of CHIT90 (NS). Biofilms were prepared using blends among Chito90 and
nanoparticles using the nanoparticles with the same distribution of degrees of polymerization
but at different NS/S(ts) ratios: 3:7, 1:1, and 7:3. The 09 different mixtures were magnetically
stirred for 5 min and subsequently individually used for the biofilm fabrication.
6.2.4 Bioplastic film fabrication
Chitosan biofilms were prepared by the knife coating method (SOUZA, H. K. S. et al.,
2013). For these purposes, 30 mL of a 3 % (w/w) solution were spread at 0.3 ms-1 onto an
acrylic plate using a film applicator (Sheen, model 1132N, UK). The plates were put in an
environmental test chamber (T= 40ºC and RH= 60 %, relative humidity) and dried for 18 h.
Subsequently, they were peeled off from their supports, rinsed with distilled water, and stored
99
for 18 h in environmental test chamber. Smooth and homogeneous films were obtained. The
film thicknesses were measured with 1 m resolution using an Absolute Digimatic Indicator
(ID-F150, Mitutoyo Co., Japan) and three independent measurements were averaged for each
case.
6.2.5 Color and Opacity
Color and opacity parameters were determined by a Minolta colorimeter CR300 series
(Tokyo, Japan). The color of the films was determined using the CIELab color parameters,
while lightness (L*) and chromaticity parameters a* (red - green) and b* (yellow – blue) were
also measured. The color of the films was expressed as the difference of color (ΔE*)
*
*
*
according to Eq.(1). The variables Ls , a s and bs are CIELab standards for the white
standard used as the film background (PENG; LI, 2014).
𝛥𝐸 ∗ = √(𝐿∗ − 𝐿∗𝑠 )2 + (𝑎∗ − 𝑎𝑠∗ )2 + (𝑏 ∗ − 𝑏𝑠∗ )2
(1)
Film opacity was determined according to the Minolta-lab method in reflectance mode
(THAKHIEW; DEVAHASTIN; SOPONRONNARIT, 2013). Opacity (Y) was calculated
from the relationship between the opacity of the film superposed on the black standard (Yblack)
and on the white standard (Ywhite), according to the following equation:
𝑌
𝑌 = 𝑌 black 100
white
(2)
For each biofilm condition, five readings were performed.
100
6.2.6 Scanning Electron Microscopy (SEM)
The SEM images were acquired in the secondary electron mode using an FEI Quanta
400 FEG microscope located at CEMUP (Centro de Materiais da Universidade do Porto).
Samples were previously cryo-fractured using liquid N2 and mounted on aluminum stubs
covered with double-coated carbon conductive adhesive tabs. Afterwards, the samples were
coated with a thin film of Au/Pb and imaged in the high-vacuum/secondary electron imaging
mode scanning electron microscope using an accelerating voltage of 10 kV and the working
distances oscillated between 10.4 to 13.8 mm.
Blend film features: mechanical resistance, water vapor permeability and water
sorption
6.2.7 Mechanical characteristics
Measurements were performed on a texture analyzer (TA.XT2, Stable Micro Systems,
Surrey, UK) equipped with tensile test attachments. The test specimens consisted of strips of
uniform width and length (25 × 100 mm) conditioned for 07 days in a climatic chamber (T=
25 ºC, RH= 53 %). Experiments were run immediately after removing the specimens from the
climatic chamber to minimize water adsorption/desorption.
The distance between the grips was 60 mm and the applied test speed was 0.1 mm s -1,
while force (N) and deformation (% strain) were recorded. Parameters such as elongation at
break (%), tensile strength (MPa), and Young’s modulus (MPa), were determined. Typical
errors in the latter parameters were 1 MPa. Five replicates were performed in each condition.
101
6.2.8 Water Vapor Permeability (WVP)
Permeability tests were conducted according to the American Society of Testing and
Materials Standard Method 96 (ASTMD882-9 1996). Transparent films, cut in 80 mm
diameter discs, were equilibrated for 7 days in the environmental chamber (T= 25ºC, RH =
53%). The films were tightly sealed in a permeation cell containing anhydrous calcium
chloride (RH = 2%) and stored at room temperature in a container filled with distilled water
(RH = 100%). Air convection was used to facilitate water diffusion. The cell was periodically
weighed and the permeability to water, given as the water vapor transfer rate (WVTR) in gms Pa-1, was calculated according to Equation (3)
1 -1
WVTR= (Δm x) / (A Δt Δp)
(3)
where Δm is the weight gain (g), x is the film thickness (m), and A is the area (0.003 m2)
exposed for a certain period Δt (s) to a partial water vapor pressure Δp (Pa).
6.2.9 Water Sorption Isotherms
The films were cut into 25 x 25 mm2 pieces and dried under vacuum at 60 ºC for 40 h.
Then, they were placed at 25 ºC into hermetic containers with equilibrium water activities
(aW) ranging between 0.1 and 0.9. The samples were periodically weighed until achieving a
constant value (2-7 days). The water sorption isotherm was determined for each sample by the
correlation of aW with the moisture content on a dry basis, Xe. The Guggenheim-Anderson-de
Boer (GAB) equation:
Xe =(C∙k∙X0∙aW) / ([1-k∙aW]∙ [1-k∙aW + C∙k∙aW])
(4)
is a typical model for fitting sigmoid-shaped equilibrium moisture isotherms in foodstuffs
(AL-MUHTASEB; MCMINN; MAGEE, 2004;
ROSA; MORAES; PINTO, 2010). X0
102
represents the monolayer moisture content, also on dry basis, and C is a constant, which
depends on the difference between the heats of sorption of the monomolecular and
multimolecular layers. k is another constant reflecting the difference between the heat of
sorption of the multilayer and the heat of water vapor condensation.
6.2.10 Statistical analysis
Data were analyzed by an ANOVA with Bonferroni’s post-test using the software
package GraphPad Prism San Diego, California, USA, version 5.00 for Windows
www.graphpad.com. The statistical significance was assessed from the standard derivation of
different measurements collected from each sample, with a significance leveled at p < 0.05.
6.3
RESULTS AND DISCUSSION
6.3.1 Mechanical properties
The determination of the mechanical properties of the chitosan biofilms, obtained by
fibres (non-sonicated (NS) sample), nanoparticles (sonicated (S) sample) and NS/S(ts)
CHIT90 blends, is essential for the adequate design of biopolymer films for packaging with a
certain degree of resistance.
Using knife coating technique, homogeneous, transparent, and flexible films (without
use of plasticizer) were obtained from NS CHIT90 mixed with various amount of chitosan
nanoparticles ranging from 6-15nm (obtained after ultrasonication of CHIT90 at 5,15 and 30
min). The influence of the nature and content of the NS, S(ts) and the NS/S(ts) mixture on the
large strain behavior of chitosan based films (conditioned at 25ºC and 53% relative humidity)
was studied up to its failure by typical tensile experiments. The mean values of tensile
strength (TS), Young’s modulus (Y) and elongation break (E) measured determined from the
corresponding stress–strain plots as well as the values of thickness (d), are displayed in Table
1.
103
Remarkably, the thickness of the film made up of non-sonicated CHIT90 (NS) was
higher to those of sonicated and NS/S(ts) blend films. The films produced by sonicated (S)
samples were more fine than the NS/S(ts) mixture. The NS (CHIT90) films presented similar
values of TS, E and Y (82.8±2.7 MPa, 2.9±0.1% and 35.2±0.9 MPa respectively. Again, times
of sonication changed the physical characteristics of the films decreasing its mechanical
properties.
Incorporation of nanoparticles into the fiber solutions leads to interesting results. In
general, films prepared with NS/S(ts) showed enhanced mechanical resistance when
compared with its sonicated precursors. Different TS behaviour was observed depending on
the films mixture preparation. In this sense an increase in TS was observed for NS/S(5 and 15
min) mixing ratio, showing a gain of resistance for the NS/S(5) 3:7 and NS/S(15) 7:3and 1:1
mixture, respectively, however a decrease in TS values was observed for other mixtures. The
NS/S(5) showed a resistance gain with the decreasing of the mixing ratio, exhibiting a better
performance when the 3:7 NS/S(5) mixing ratio was used. Regarding the film formulation
NS/S(30) mixing ratio, no variation was observed in the maximum film stress of films (Table
1).
The film ability to stretch (E) also varied for the different film formulations (Table 1).
When compared the S(5) films, an improvement of the E values was verified at the different
mixtures, if they are compared with its sonicated precursors. As it can be observed, an
increment in nanoparticles in the mixture leads to an increase in E properties for NS/S(5)
mixing ratio. Nevertheless, the increasing in nanoparticles ratios seems not to influence the E
values for the remaining film formulations NS/S(15 and 30) mixing ratios. ,the different
behavior observed for the blended NS/S(ts) films may be ascribed to some balance of the
mechanical properties, thanks to the presence of the microfibers, provided by the NPs.
The film that presented the highest TS and E was those prepared with 7:3 (95.9±3.5
and 5.3±3.0) respectively and 1:1 (96.1±3.1 and 6.9±0.9 respectively) ratio, having a higher
value than NS CHIT90 film and two times greater than the precursors S(15). From these
results, it is possible to infer that the better performance of these films seems to be due to the
size of the nanoparticles obtained after 15 min sonication. Those blends contain larger and
adequate number of β→1,4 glycoside bonds and a high number of active sites at the end of
polymer chains (GOCHO et al., 2000) than that S(5), which enables a higher interaction
between the molecules, making them very strong. Additionally, the polymers chains in the
104
blended film are tightly bounded, with a decrease in the degree of flexibility for NS films and
an increase for the nanoparticled ones.
On the other hand, the NS/S(5) mixing ratio, 1:1 film showed a remarkable
improvement of the percentage of elongation (E%), fourfold higher than NS sample, and six
times higher than nanoparticle pioneers S(5) film. The nanoparticles used to make this film
has a larger size than those produced by the ultrasound treatments for 15 and 30 min (Capítulo
III), allowing for a large number of crosslinks that increase the elongation at break feature. It
seems that the polymer chains of the blended film, although covalently bounded, preserved a
certain degree of chain flexibility.
Regarding the elastic modulus (Y), three groups with distinct properties were
observed: (i) films prepared with the NS/S(5) mixture presented lower values of Y than their
NS and S precursors; (ii) an opposite behavior was observed for films prepared with NS/S(15)
and (iii) NS/S(30) films presented values of Y lower than NS films and threefold higher than
the S(30) films. However, within each group, an increase of nanoparticles in the blend
formulation does not cause differences in Y.
The nanoparticles generated by ultrasound treatments produced new interactions by
crosslinking through the chitosan amino group that would normally be involved in the
hydrogen binding between the chitosan fibers (CHIT90) and the nanoparticles (ALBANNA et
al., 2013; OSTROWSKA-CZUBENKO; GIERSZEWSKA-DRUŻYŃSKA, 2009) In films
made up of chitosan and alginate, it was demonstrated that a three-dimensional network was
formed by chemical or physical crosslinking of the hydrophilic polymer chains
(OSTROWSKA-CZUBENKO; GIERSZEWSKA-DRUŻYŃSKA, 2009). In chemical gels,
polymer chains are connected by covalent bonds, but in physical gels they are held together
by molecular entanglements, not covalently bonds, such as Van der Waals interactions, ionic
interactions, hydrogen bonding, hydrophobic interactions, traces of crystallinity and multiple
helices.
Mechanical testing results confirmed the proposed hypothesis that mixing fiber and
chitosan nanoparticles results in an improvement film in improved TS, E and Y films
properties. In this sense, in the NS/S(ts) chitosan films presented here, the nanoparticles seem
to have major contact with the surfaces due to their smaller size, increasing the number of
chemical crosslinks with the biofilm matrix (CHIT90), thus being able to make more
connections and, consequently, improving the mechanical properties of these films.
105
Usually, all mixtures used produced fiber/nanoparticle films with improved strength
and stiffness and decreased fiber elasticity to different extents when compared with NS.
Without use of any plasticizer, that could be transfered to the packaged food, these
improvements in fiber/nanoparticles film properties should broaden the utility to fabrication
of biofilms to be regularly used in agro-resources and in biotechnology as a substitute of biofuel plastic.
106
Table 1. Film thickness (d), tensile properties (tensile strength, TS; elongation at break, E; and Young´s modulus, Y) and water vapor
permeability (WVP) for solid chitosan films prepared from Chito90 and nanoparticle mixtures.
CHIT90
NS
NS/S(5 min)
Mixing
ratio
TS / MPa
E/%
Y / MPa
0
0.01±0.007a
82.8±2.7ª,b
2.9±0.1a,b
35.2±0.9a,b,c
14.2±1.1
7:3
0.023±0.004b,c
67.3±3.7b,c
5.1±0.4c
16.1±4.3d,h,i
7.89±0.11
1:1
0.025±0.005b
64.3±7.6b,c
12.9±0.8
16.2±1.0d,h
9.46 ±4.5
3:7
56.0±9.5c,d
17.6±2.3
2.67±0.7ª,b
18.0±0.8d,e,f
c,d,e
b,k
1:1
0.017±0.003c,d,g
96.1±3.1e
6.94±0.9e
50.0±9.4k
b,d,e,f
c
e
a
0.022±0.005
0.008±0.001a
7:3
0.027±0.01
f
c,e
95.9±3.5
e
9.58±0.6
d,e,g
0.019±0.001
S(15 min)
S(30 min)
b,d
100.6±10.5
a,e
7:3
3:7
NS/S(30 min)
0.019±0.002
b,c
0.008±0.006a
S(5 min)
NS/S(15 min)
WVP∙10-11/ g.m-1s1
Pa-1
d / mm
68.9±3.2
61.1±20.0c,f
34.1±8.4
49.7±3.0
h,i
d,f,i
5.3±3.0
6.86±1.4
3.5±0.1d
1.51±0.3
b
1.86±0.16
a,b
40.7±1.5
37.9±5.4
29.5±5.0a,b,c,e,j
24.6±11.2
5.92 ±0.72
13.6±4.4
13.2 ±3.23
12.1 ±0.15
9.08 ±0.7
7.8±2.0
f,g,i,j
38.9±7.9
8.70 ±5.3
a,k
1:1
0.018±0.003
3:7
0.011±0.001a,g
41.0±1.1d,g,h
3.0±1.0a,b
29.2±3.3c,f
3. 93 ±0.41,2,4
0.009±0.003a
26.5±7.9g
2.72±.0.9b,a
9.1±1.3g,h
7.9±0.7
6.27 ±3.91,2,4
Values are given as means ± standard deviations. Same subscript letters within the same column indicate non-significant differences (p >
0.05, Bonferroni test). For WVP parameter, the samples with the same number showed significant differences, number 1 represent
difference for sample NS; number 1 represent difference for sample NS; number 2 represent difference for sample S(5 min); number 3
represent difference for sample NS(5)3:7; number 4 represent difference for sample NS(15)7:3, (p < 0.05, Bonferroni test).
107
6.3.2 Water vapor permeability (WVP) and moisture sorption isotherm
The water permeability and water absorption properties of films are involved with the
diffusion of molecules through the film matrix, representing the equilibrium relationship
between its moisture content and the water activity (aw), which are necessary to predict the
properties of films in different environments.
In line with this discussion, the water vapour permeability measures of the NS, S and
NS/S(ts) of the films are displayed in Table 1. Films produced with sonicated chitosan seem
to decrease water vapor permeability (WVP), while maintaining other desirable mechanical
properties. In other words, when compared with NS WVP values, the blends obtained with
sonicated nanoparticles NS/S(ts), showed decreased: water permeating per area unit area
(WVP) and time to through the packing material. The WVP values ranged between 3.9 – 14.2
x 10-11/g.m-1.s-1.Pa-1 (Table 1).
The chitosan film reinforced with S (15 min) chitosan nanoparticles had the highest
WVP, ranging from 13.2 – 9.08.x 10-11/g.m-1.s-1.Pa-1. This was because chitosan nanoparticle
obtained after 15 min sonication decreased the compactness of the films. The blend films
made with S(30) showed the smallest WVP values, which decreased as the proportion of
nanoparticles was enhanced. Values varied between 3.9 – 8.7 x 10-11/g.m-1.s-1.Pa-1 a decrease
is WVP was observed in the 3:7 mixing (table 1). The behaviour could be explained since the
lower diffusion rates for water or gas molecules result from transport obstruction through the
molecular network packing. (RAMOS et al., 2013). In general, the WVP values of NS
chitosan films are similar to that found for chitosan films prepared without plasticizers
(HWANG et al., 2003; SOUZA, H. K. S. et al., 2013) . However these values are lower when
compared with those made up by chitosan DD 92% solution diluted in acetic acid (KIM, K.
M. et al., 2006) and by other biopolymers, such as protein and cellulose (PARK; CHINNAN,
1995).
In a recent study (SOUZA, H. K. S. et al., 2013), it was demonstrated that films
prepared from the most ruptured degraded CHIT90 exhibited the lowest water permeability.
Small and spherical low molecular mass chitosan particles contain larger spherulytic
crystallite and more crystalline domains, which enhances the resistance to solvent penetration
(SOUZA, B. W. S. et al., 2010).
108
The WVP results found here are in agreement with this hypothesis. Since, the 3:7 ratio
NS/S(30) films exhibited lowest WVP values, it can hypothesize that an increase in S(30)
nanoparticle proportion result in WVP decreases, confirming that these samples filled the
voids shaped by the fibril matrix and consequently result in films where the permeation of
water molecules is more difficult. However, when chitosan nanoparticles were obtained after
15 min sonication, it seems that the nanoparticles would tend to form aggregates, which could
not contribute to preventing water molecule diffusion; thus, the WVP is increased.
Additional increment of chitosan nanoparticles induced a reduction in WVP for others
systems. For instance, chitosan nanoparticles increased the barrier effect for banana films
(FABER et al., 2004), in poly(caprolactone), composite films (FABER et al., 2005) and was
successfully used as reinforcement material in methylcelluose based film (DE MOURA et al.,
2009). Since these features are involved in the physicochemical and microbiological
deterioration of food (JANJARASSKUL; KROCHTA, 2010), the NS/S blends obtained
herein show desirable characteristic for biofilms to be applied on fruit and seed coating.
Moistures of the sorption isotherms are important to evaluate the effect of temperature
and relative humidity on film properties (LAI; PADUA, 1998). Addition or removal of water
may cause phase transitions in the macromolecular structure (TORRES, 1994). The GAB was
used to fit the water adsorption data of the NS, S and NS/S(ts) films. A goodness of the fit
was acquired and the fitting parameters are displayed in Table 2.
The change in the moisture sorption isotherms of NS, S(ts) and NS/S(ts) showed the
equilibrium moisture content of all of these films was dramatically increased in values above
aw = 0.6 for precursor (NS and S(ts)) films and aw = 0.3 for the blends (NS/S(ts)), was
observed the presence of nanoparticles affected the moisture sorption isotherms of the
NS/S(ts) films (figures 1).
The equilibrium moisture content of the NS/S(ts) films decreased as nanoparticles is
submitted to different time period of sonication (ts= 5, 15 or 30 min). The NS/S(30) presents
the lowest moisture content at a given aw. It seems that the smallest nanoparticles provide less
active sites to bind water molecules hydroxyl groups.
The isothermal results of NS and S(ts) films showed good agreement with those
previously described (SOUZA, H. K. S. et al., 2013). The k values are in accordance with the
recommended values (0.6<k<1.0) (GARCıA
́ -OLMEDO et al., 2001). The amount of water
109
involved in the adsorption of one monolayer is quantitatively described (on a dry basis) by X 0
(SOUZA, H. K. S. et al., 2013). In the present study, the blend films showed different
behaviour, according to the qualitative analysis of the curves, an increase of X0 values is
observed for NS/S 15 and 30 min mixtures when compared with sonicated precursors. X0
also was higher for these mixtures, indicating that these films present the highest number of
sorption sites that adsorbed in a monolayer of dry films. No significant differences in X0 were
observed at distinct NS/S mixing ratios.
Table 2. GAB parameters obtained from fitting the data in the GAB model. Values are
displayed as mean ± standard deviations.
Chit 90
Mix ratio
C
k
Xo
R2
NS
NS/S (5 min)
0
7:3
1:1
3:7
0,017
2.84
1.6
1,0
2.0
5.5
3,5
2.2
3.1
0,9
1.0
1.0
1.0
0.90
0.99
0.88
0.95
1.05
0,12
0,0045
0.01
0,01
0.01
0.1
0,008
0.1
0.003
0,9908
0,9813
0.991
0,9818
0.997
0.995
0.995
0.999
0.997
7:3
0,0023
1,0
0,1
0,99289
1:1
3:7
0,0023
0,0136
11.6
1,0
0,9
1.0
0.1
0.1
0.002
0,98445
0,97354
0.998
S (5 min)
NS/S (15 min)
S (15 min)
NS/S (30 min)
Mixing ratio
S (30 min)
7:3
1:1
3:7
Chitosan has three predominant adsorption sites: the hydroxyl group, the amino group
and the polymer chain ends. The decrease in molecular weight increases the number of
polymer chain ends per unit weight. Thereby, the adsorption sites are newly produced from
increasing degradation process, due to the decrease in the molecular weight of chitosan
(GOCHO et al., 2000). This observation is in agreement with mechanical properties results
because of the highest young modulus values that was found in NS/S(15 and 30) mixtures.
Instead to the data observed for the set of blend films containing S(5 min) showed a decrease
in water activity (aw) in one monolayer adsorption, where it was observed lower values of Xo,
110
representing low number of sorption sites in a monolayer of dry films. The values of Xo for
these samples are similar considering all ratios used.
Accompanied by k values approaching to 1, a very high value of C was found for
NS/S(15) in the blend, 7:3. This kind of GAB parameter combinations is related to the
multilayer molecules of the film, which have properties comparable with those of bulk liquid
molecules. In other words, high C values in the blend film could be associated with the
sorption of water by NS/S(15) 7:3 ratio, that is apparently characterized by a monolayer of
molecules. The following molecules are a little structured (or not) in a multilayer,
nevertheless its characteristics are comparable with the molecules in bulk liquid.
In contrast the NS/S(30) showed decreased C values. The water molecules are less
strongly bounded to the material since they are organized in monolayer. These materials are
also organized in multilayers where upon the water molecules differ significantly from bulk
liquid molecules. Considering also that the smallest nanoparticles (30 min) provide a petty
number of adsorption sites, and present high water adsorption (X0), one can conclude that
high and strong interactions could occur between fibres and nanoparticles. These results are in
line with the WVP measures. As discussed above the, blend prepared with 3:7 NS/S(30)
presented the lowest WVP values and, on the contrary, the NS/S(15) blend was considered the
most permeable.
Additionally, X0 and C values was lower when compared with NS/S(5) and NS/S(15)
blends. This behaviour indicates that NS/S(5) adsorbed less water than the NS/S(15).
However, NS/S(5) blends presented C values higher than NS/S(30), showing more adsorption
of water for these samples. Again these results corroborate with WVP measures where WVP
values for NS/S(5) samples fluctuated between NS/S(15 and 30) levels.
Sorption results are also in accordance with mechanical properties results. Since
nanoparticles provided after 15 min of sonication are larger than those obtained after 30 min.
The former samples presented more β→1,4 linked. When mixed with NS samples generated
films with a superior resistance (stronger bonds, see Table 1). However they present high C
and WVP values due to the not homogeneous filling of fibre holes. Regarding NS/S(30), the
sample presented the lowest TS (less β→1,4 linkage and smaller macromolecules) and C
values. In this case, nanoparticles (S) are more cohesively structured with the fibres (NS)
resulting in homogeneous filling (closing the holes that exist in fibre structures) not leaving
111
spaces for water molecules which make these films less permeable and more compacted
(figure 3).
112
Figure 1. Effect of different NS/S(ts) mixing ratio concentrations on the equilibrium moisture content of
the films, fitted with GAB model. The GAB fitting (solid lines, for each sample), NS (green stars, all panels)
and S (pink triangles, all panels)., For each system the following NS/S ratios were studied: 7:3 (black squares),
1:1 (red circles), and 3:7 (blue triangles) in all panels;. Sonicated for ts =5 (panel A), 15 (panel B), and 30 min
(panel C).
113
6.3.3 Morphology of the chitosan blend films
Cross sections of the cryo-fractured NS (CHIT90) and S (5 min) are showed in Fig 2A
and B, and 1:1 blends to NS/S (5, 15 and 30) are presented in Figs 2. In line with the
previous results NS and S(5) present large fibers and spherical nanoparticles, respectively.
Figure 2. Representative SEM pictures of the cross-sections of cryo-fractured films at 30,000x magnification (A
— NS CHIT90 and B — S(5).
114
The SEM micrographs of the fractured surface of NS/S (5, 15 and 30 min) of 1:1
blends films are illustrated in Figure 3. It was observed, a distinct superficial arrangement
after the incorporation of nanoparticles into the fiber film matrixes resulting in films with a
large number of small discrete nanoparticles within fibres as exhibited in the Fig 3, and more
clearly showed in the Fig 3A.
As observed in a previous study (Capítulo II) the size of nanoparticles are dependent
of sonication time sonication. For instance, image of blends prepared at 1:1 ratio with S(15
and 30) (Figure 3B and 3C) showed a cross-section more homogeneous and smooth. This
observation shows that after adding the nanoparticles sonicated by 15 and 30 min the
interfacial interaction between fibers from NS chitosan and S were improved.
These results are in accordance with mechanical and WVP evaluations. As discussed
above, a very good improvement of mechanical properties (TS, E and Y) was observed for
NS/S(15) 1:1 when compared with their precursors. Nevertheless blends prepared with NS/S
(1:1) 30 min presented also an improvement of mechanical properties (when compared with
their sonicated precursors), showing the lowest WVP values.
115
Figure 3. Representative SEM pictures of the cross-sections of cryo-fractured NS/S(ts) 1:1 ratio films at 30,000x
magnification A — NS/S(5), B — NS/S(15) and C— NS/S(30).Red arrows indicated the preference of small
fibrils and spherical nanoparticles, apparently distributed within the film.
116
6.3.4 Color properties
Color is an important object measurement for image understanding and object
description, used for conferring quality evaluation and inspection of food products. Color
measurements can be performed by visual (human) inspection, traditional instruments such as
colorimeters, or computer vision (WU; SUN, 2013). The parameter ΔE provides a good
measure of color difference, since it takes in consideration the following three parameters:
lightness (L), red – green (a) and yellow – blue (b) components.
The total color difference and opacity of the films produced are summarized in Table
3. All blends of chitosan films were very transparent, with L coordinate ≥ 91. When color
parameters of blend films were compared to those CHIT90 films, significant differences were
observed when 15 and 30 min-sonicated particles were used. Addition of nanoparticles into
the fibers affected the a* (red-green) and the b* (yellow-blue) measurements. Great values of
b* were observed for NS/S (15 min).
117
Table 3. Color parameters
CHIT90
NS
NS/S(5
min)
Mixing
ratio
L*
a*
b*
ΔE*
Opacity
0
95.67±0.2a
-0.302±0.04a
3.26±0.2a
2.1±0.3a
10.4±0.2a
97.22±0.2c
-0.21±0.1a
3.05±0.8a,b,d
1.3±0.8a
-
1:1
95.38±0.1a
-0.27±0.06a
3.4±0.2c,d
2.4±0.2a
12.2±1.9a,b
3:7
96.88±0.8b,c
-0.33±0.3a
3.9±1.7a,c
2.3±1.8a
-
96.17±0.5a,b
0.002±0.008a
2.6±0.1a,b
1.3±0.4a
12.3±0.8b
91.30±0.8e
-0.53±0.06a
5.52±0.2e
7.0±0.7c
13.4±0.6c
1:1
92.65±0.8d
-0.52±0.07a
5.05±0.2e
5.6±0.7b
13.2±0.4c
3:7
95.24±1.0a
-0.26±0.04a
3.72±0.2a
2.8±0.8a
10.4±1.5a
96.25±0.4a
0.07±0.04a
2.57±0.3e
1.8±0.5a
12.3±0.7c
95.01±0.6i
-0.11±0.1a
3.39±0.4a,g
2.6±0.8a,e
10.7±0.9a,e
1:1
95.86±0.7a,h
-0.18±0.08a
3.42±0.2a,g
2.1±0.5a,e
12.2±1.1d,e
3:7
94.63±0.7g
-0.32±0.04a
3.90±0.3g
3.3±0.7e
12.0±1,1a,d,e
93.34±0.6f
-0.56±0.06a
5.2±0.3f
5.1±0.7d
12.6±0.6d
7:3
S(5 min)
NS/S(15
min)
7:3
S(15 min)
NS/S(30
min)
7:3
S(30 min)
CIELab standards were used as the white standard with the film background: Ls*(97.10), as*(0.05) and
bs*(1.76). Mean values ± standard deviations. Superscripts in the same column with different letters indicate
significant differences (p < 0.05).
Sonication time periods lead to a significant effect on the total color differences when
compared withNS blend films. The S(30) films presented the highest value of ∆E (5.1 ±0.7).
Regarding mixture films, incorporation of nanoparticles (S:15 and 30 min) had a significant
increased effect on the ∆E, that is, the high S(15), the highest ∆E for those blend films in the
films 7:3 and 1:1 ratio. Additionally, the results indicated that the addition of S(5) to the blend
does not lead to statistically significant changes (p > 0.05) in ∆E values . The highest values
of opacity was founded in NS/S(15) 7:3 and 1:1 ratios.
118
The total color difference (ΔE) observed between all samples was discrete in
comparison with other films, mainly those produced from soybean protein concentrate with
plasticizer (CIANNAMEA; STEFANI; RUSECKAITE, 2014). Indeed, it has considered that
the incorporation of plasticizers can promote more transparency, since these molecules
interact between protein chains, decreasing the absorbance in the visible region
(SOTHORNVIT, R. et al., 2007).
Even in the chitosan films, the addition of plasticizers such as glycerol and Tween 20
showed L value that indicates film lightness and yellowness decreases (ZIANI et al., 2008) as
reported previously (LOPEZ et al., 2014).
An increment of nanoparticles in the blend led to an increase in the films’ opacity
comparatively with NS film (table 4). The blend films containing S 15 min showed the
highest opacity. The nanoparticles may be embedded in the interspaces of chitosan film,
which prevented light transmittance and leading to higher opacity.
Table 4 shows that, in general, with the addition of nanoparticles, the opacity value of
the NS/S(ts) films increase when compared with NS film , suggesting that the chitosan fiber
film was more transparent than the NS/S films. Highest values of opacity was found in
NS/S(15) 7:3 and 1:1 ratio.
6.4
CONCLUSION
In this study it was produced fine films by knife-coating technic, from blends of nonsonicated (CHIT90) and colloidal chitosans, NS/S(ts) (3:7, 1:1 and 7:3). The colloidal
solutions produced by ultrassonication (for different times ts= 5, 15 and 30min), present
various morphologies and molecular weights. The results herein showed that it was produced
films with different mechanical, water moister proprieties and WVP adjusted though the
controlling of mixing ratios of NS/S(ts) it is able to being able to produce flexible films with
good transparency and homogeneous morphology (without use of plasticizer).
The film produced with proportion NS/S(5) 3:7and NS/S (15) 7:3 e 1:1,showed better
tense average. The increment in nanoparticles leads to an increase in E properties for NS/S(5
and 15 min) mixing ratios. Chitosan film reinforced with S(ts) showed an improvement at the
WVP. Additional increment of chitosan nanoparticles induced a reduction of WVP for others
systems, the blends made with S(30) showed the smallest WVP values. The equilibrium
119
moisture content of all films dramatically increased above aw = 0.6 for precursors films and
aw = 0.3 for the blends. Films reinforced with nanoparticles showed little change in the color
proprieties without compromised the film transparencies.
120
7
ANTIMICROBIAL ACTIVITY OF CHITOSAN NANOPARTICLES OBTAINED BY
ULTRASOUND IRRADIATION (Manuscrito em preparação)
Abstract
Chitosan is a natural polysaccharide prepared by the N-deacetylation of chitin. In this study,
the physicalchemical and inhibitory characteristics of chitosan nanoparticles produced by
ultrasound irradiation were evaluated. The physicochemical properties of the nanoparticles
were determined by dynamic light scattering and zeta potential analysis. The antibacterial
activity of chitosan nanoparticles against E. coli was evaluated by calculation of minimum
inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Results
showed that chitosan nanoparticles could inhibit the growth of the bacteria tested. The MIC
values were inferior to 0.5mg/mL, and the MBC values of nanoparticles were similar or
higher than MIC. DNA release measurements revealed that the exposure of E. coli to the
chitosan nanoparticles led to the disruption of cell membranes and the leakage of cytoplasm
and nucleus contents.
7.1
INTRODUCTION
During the last decades, the number of studies on the use of engineered nanoparticles
(NPs) and nanomaterials (NMs) has increased in many research areas, with important
applications in water purification, drug delivery, antimicrobial treatments, tissue engineering,
biosensors, detection of analyses related to magnetic diagnostics and detection: pathogen,
toxins, drug residues and vitamins (THATAI et al., 2014). Several methods have been
developed for the production of NPs, such as ionotropic gelation, spray drying,
emulsification, coacervation and, more recently, ultrassonication (SINHA et al., 2004) One of
the applications of high intensity ultrasound (US) is the effective polymer degradation,
breaking up aggregates and reducing NP size and polydispersity. Ultrassonication leads to the
decrease in the mean diameter and polydispersity of particle size when the duration time or
amplitude of the irradiation is increased (GOMES, LAIDSON P. et al., 2016; TANG, E. S.
K. et al., 2003). The application of studies regarding nano-based composites and the physical
stability of NPs are directly linked to material characteristics, such as chemical composition,
size, shape, surface charge density, hydrophobicity (NEL et al., 2006), polydispersity and the
presence or absence of functional groups or other chemicals (MAGREZ et al., 2006). These
important characteristics define the potential applications for NPs.
Biodegradable NPs have being attracted great attention, due to their ability not only to
protect proteins and peptides from degradation but also due to their desirable release profiles.
121
Among these macromolecules, chitosan (CS) has been studied during the last two decades.
CS is produced by the removal of acetyl groups by alkali or enzymatic (GOMES, LAIDSON
P. et al., 2014) treatment of the chitin chain leaving behind a reactive amino group (NH2),
responsible for chitosan versatility (WANG, W. et al., 2008). As a natural polysaccharide and
potential biological compatibility with chemically versatile NH3+ groups, the amino groups, at
pH lower than 6.5 the amines are protonated and CS acquires polycationic behavior, showed a
polyelectrolyte behavior (NAGPAL; SINGH; MISHRA, 2010).
Chitosan possesses a wide range of useful properties, specifically; biocompatible,
biodegradability and antibacterial activity. CS applications include water treatment,
chromatography, cosmetic additives, coatings for food conservation, as fruits and sliced
sausages, seed preservation against microbial spoilage, production of biodegradable films and
microcapsule implants for drug and vaccine delivery (Capítulo I) (XIA et al., 2011). The
inhibition activity described for chitosan has been observed that depends on its molecular
wight (MW), degree acetylation (DA), pH and, of course, the target organism
(HERNÁNDEZ-LAUZARDO et al., 2008;
TSAI; SU, 1999). Chitosan shows several
advantages over other types of disinfectants, since it possesses high-antimicrobial activity, a
broad activity spectrum and low toxicity to mammalian cells (LIU, X. D. et al., 2001).
Nanoscale chitosan and its derivatives have also been considered as antimicrobial
agent against bacteria, viruses and fungi (QI et al., 2004) The nano-CS may present several
molecular weights and show known antimicrobial activity, some studies demonstrated low
toxicity, being safe for human applications (MUZZARELLI; A, 2011).
The investigation of chitosan antimicrobial properties has been a long journey of
scientific exploration and technological development. The distinct physical states and MWs of
chitosan and its derivatives render distinctive antibacterial action mechanisms. Chitosan can
inhibit and suppress microbial activities through electrostatic charge interaction between the
positive charges on polycationic chitosan macromolecules (amino groups) with the negative
charges on the microbial surface (AZIZ et al., 2012). This interaction causes disruption on the
microbial cells, which then change their metabolism and lead to cell death (EL-SHARIF;
HUSSAIN, 2011; LECETA et al., 2013). On the other hand, low molecular weight watersoluble chitosan and ultrafine nanoparticles can penetrate bacterial cell walls and combine
with bacterial DNA, inhibiting mRNA synthesis and DNA transcription (SUDARSHAN, N.
R. et al., 1992). HMW water-soluble chitosan and solid chitosan, including larger sized
nanoparticles, interact with the cell surface instead, altering cell permeability (CHOI et al.,
122
2001; EATON et al., 2008; LEUBA; STOSSEL, 1986), resulting in membrane degradation
or forming an impermeable layer around the cell, at blocks the transport of essential solutes
into the cell .
Although, there are few studies on NPs forms of CS concerning to antimicrobial
activity, the small size of chitosan nanoparticles renders them unique physicochemical
properties, such as large surface area (providing more cationic sites) and high reactivity,
which could, thus, potentially enhance the charge interaction on the microbial surface and
lead to superior antimicrobial effects (ZHANG, L. et al., 2010).
The objective of this study is to evaluate the physicalchemical characteristics of
chitosan NPs produced by green process chemistry and its antimicrobial activity against food
pathogens as Escherichia coli and Staphylococcus aureus process evaluate from antimicrobial
activity.
7.2 MATERIALS AND METHODS
7.2.1 Polymer degradation
Nanoparticles (NPs) were produced from two commercial chitosan, of medium (CSMMW) and low molecular weight (CS-LMW) with degree acetylation >75% (SigmaAldrich® Saint-Louis, MO - USA). Samples (2%) were solubilized in 0.1 M sodium acetate
pH 4.0, in ice bath and irradiated with ultrasonic probe SONIC model 750 W, equipped with a
1/2" microtip at 4ºC for 30 min. The irradiation was performed under constant duty cycle and
40% amplitude with 1/1s intervals.
7.2.2. Dynamic light scattering (DLS) measurements
The measurements of hydrodynamic radius (Rh) and polydispersity index (PdI) of, pre
(CS-MMW and CS-LMW) and post (CS-MMW30 and CS-LMW30) irradiation, were
performed using a DynaPro NanoStar (Wyatt Technology Corporation, Santa Barbara, USA).
123
7.2.3 Zeta potential (ζ-potential)
Zeta potential measurements were performed by applying an electric field across the
samples and the value of the zeta potential was obtained by measuring the velocity of the
electrophoretic mobility of the particles using the laser Doppler anemometry technique. The
measurements were performed five times for each sample at 25 °C using a NanoBrook 90Plus
PALS (Brookhaven Instruments Corporation, New York, USA).
7.2.4 Antimicrobial activity
7.2.4.1 Determination of minimum inhibitory concentration (MIC)
Escherichia coli DH5α strain (INCQS-Fiocruz) was grown in Luria-Bertani (LB)
medium (BD, Sparks Glencoe, EUA), in an orbital shaker at 200 rpm, 37°C, for 24 h. One
milliliter of the culture was centrifuged at 5000 x g for 2 min and the supernatant was
discarded. The cellular density was adjusted in saline solution (0.85% NaCl) to the turbidity
equivalent to McFarland 0.5 standard (1.5x108 CFU/ml) (SHUKLA, SUDHEESH K et al.,
2013) and it was used as an inoculum. The antimicrobial activity of the produced chitosan
samples (CS-MMW, CS-LMW, CS-MMW30 and CS-LMW30) were evaluated on batch
cultures containing chitosan derivatives 0.5 and1.0 mg/ml in suspension against 107 CFU/ml.
One hundred microliters of the each mixture was withdrawn and added to 96 wells microplate
(Cellstar, Greiner bio). The microplates were incubated at the same conditions above for 24 h
and the optical densities were determined at 620 nm by VitorX4 (PerkinElmer, USA). The
inhibitory percentage was calculated using the method described by MADUREIRA et al.
(2015).
124
7.2.4.2 Determination of the minimal bacterial concentration (MBC)
The MBC concentration that allows the initial viable cells to be reduced at least 99.9%
was evaluated by adding 20 μl aliquots of negative wells (no growth), plated on LB and
incubated at 37 °C for 48 h.
7.2.4.3 In vitro growth inhibition of chitosan
Antibacterial activity of chitosan against E. coli DH5α was evaluated by inoculating
100 µL overnight bacterial culture at 37 °C with 220 rpm into chitosan solutions of 0.5 and
1.0 mg/mL, . In addition, the effect of the incubation time was evaluated by inoculating
100µL of the overnight bacteria culture into chitosan solution and incubating the mixture for
1, 2, 4 and 6 h at 37°C. Bacteria enumeration was determined by counting of colonies on LB
plate after overnight at 37 °C.. The experiments were performed in triplicate.
7.2.5 Integrity of cell membrane
Cell membrane integrity of E. coli was as reported in the literature (SUDARSHAN, N.;
HOOVER, D.; KNORR, D., 1992). In brief, the cultured bacteria were harvested in the
different intervals of time (1, 2, 4, 6 h), washed twice, and suspended in sterile 0.85% NaCl
solution. The bacterial suspension was adjusted to 0.6 at OD630nm and the absorbance at 260
nm was accompanied by spectrophotometry (Beckman DU 640 spectrophotometer,
USA).Chitosan stock was inoculated into bacterial suspension to give a final concentration of
chitosan at 0.5 and 1mg/mL, respectively, during a same period of time of incubations
described above.
125
7.3 RESULTS AND DISCUSSION
7.3.1 DLS and ζ-potential
The particle size distributions were calculated by DLS and ζ-potential measures before
(CS-MMW and CS-LMW) and after (CS-MMW30 and CS-LMW30) the ultrassonication for
30 min. The chitosan fragmentation process by ultrasound and its capacity to produce
nanoparticles is already known (Capítulo III), was show in Table 1. The irradiation process
increased the % intensity of smaller macromolecules obtained from the degradation of
commercial chitosan. The high range of the polydispersity index, 2- 4% (PdI), is an indicative
of the existence of group of particles with different sizes (ARORA; PADUA, 2010).
The radius mean values of measured at 25 °C achieved shown table 1. The radius of
chitosans ranged from 219 nm to 3083 nm. The ultrasonic irradiation resulted in the increase
of the ratio of macromolecules with low Rh. The CS-LMW sample showed a major
proportion of macromolecules with Rh of 3007 nm (72%) and a smaller proportion of 425 nm
macromolecules Rh (18%), after ultrasound. The ultrasonic irradiation of CS-LMW chitosan
by 30 min generate the CS-LMW-30 macromolecules with 492 nm and 2794 nm Rh values,
with intensity of 93 and 26%, respectively. The CS-MMW is composed macromolecules of
Rh values between 403 and 3083 nm with intensity of 47 and 31%, respectively. After the
ultrasound irradiation for 30 min, it was yielded the CS-MMW-30 sample containing small
macromolecules with Rh ranging from 218 to 482 nm with intensity of 67 and 28%,
respectively.
Table 1. Hydrodynamic radius (Rh), intensity percentage of light scattering (% Intensity), polydispersity index
(% PdI) measures by DLS and zeta potential (ζ) of chitosan samples.
Sample
CS-LMW
CS-LMW-30
CS-MMW
CS-MMW-30
ζ potential (mV)
24.51±1.29
26.12±0.85
26.52±2.4
24.78±2.4
Radius (nm)
425
3007
492
2794
403
3083
219
482
% Intensity
18.8
71.9
93.1
26.4
46.9
31.8
67.0
28.8
% PdI
4.49
4.24
1.83
2.88
3.4
1.8
3.3
3.8
The chitosan samples measures before (shaded column) and after irradiation (non-shaded column).
126
The ζ-potential represents the electrical potential of solutions and can represent the
particle stability influence in suspension, through the electrostatic repulsion between particles.
When the ζ-potential is high (close 30 mV), the suspension is more stable avoid particles
agglomeration, due a higher electrostatic repulsion when comparing suspensions with lower ζpotential values. (ARORA; PADUA, 2010)
7.3.2 MIC and MBC determination
The antimicrobial activity potential of both fibrils and ultrasound treated nanoparticles
was evaluated. The MIC and MBC for the pre (CS-MMW e CS-LMW) and the post (CSMMW30 e CS-LMW30) irradiated chitosan samples were evaluated against E. coli. It was
also evaluated the influence of pH of the culture medium at the inhibitory activities (Table 2).
The MBC values are similar or higher than MIC ones. The chitosan concentrations used
to kill and inhibit bacteria are very close. The chitosans, pre and post sonicated samples
showed bactericidal behavior, acting (i) by the lysis of cell wall and/or cell membrane of
target bacteria or (ii) by inhibiting DNA or RNA, injuring the replication nuclear process
and/or gene transcription (YANG, C. et al., 2014). MIC values are in the range of 0.2 and 0.4
mg/mL, depending on pH of medium. The sample that showed to be more effective was CSLMW30 and the less effective ones were CS-MMW and CS-MM30. The superior activity of
CS-LMW and CS-LMW30 corroborate data from literature, where the chitosan activity is
dependent on MW and DA (PATEL, J.; JIVANI, 2009).
Chitosans show low solubility in few weak environments as sodium acetate, pKa near
6.0. When pKa values are higher, the solubility is decreased, and may occur polymers
precipitation. At pH just below pKa, chitosan shows polyelectrolytic behavior because of its
charged amine radical (CÖLFEN et al., 2001). The protonation of chitosan cause it dispersion
and turn it more effective against E. coli. MIC values against E. coli showed pH dependent,
for CS-MMW (0.47 and 0.4) CS-MMW30 (0.4 and 0.28) and for CS-LMW (0.3 and 0.33)
and CS-LMW30 (0.3 and 0.2), for pH 7.0 and 5.0 respectively.
The results showed here demonstrated that the production of nanoparticles by
modification of the polymer chain length by ultrasound irradiation is a simple method and
127
dispersed the addition of acid solvent. The MW modifications provoke satisfactory changes in
the antimicrobial potential of the nanoparticles produced.
Table 2. MIC and MBC from different chitosan samples against E. coli cells. Pre-sonicated, medium
molecular mass (CS-MMW) and low molecular mass chitosans (CS-LMW) and post-sonicated medium
molecular mass (CS-MMW30) and low molecular mass (CS-LMW30) chitosan samples had the MIC and MBC
measured against E. coli (DH5).
Sample
MIC (mg/mL)
pH 7.0
CS-MMW
CS-MMW30
CS-LMW
CS-LMW30
0.47±0.06
0.4±0.00
a
a
0.3±0.00b
MBC (mg/mL)
pH 5.0
0.4±0.00a
0.28±0.03b,c
0.33±0.06
pH 7.0
0.4
0.4
c
0.3
pH 5.0
0.4
0.3
0.3
0.3±0.00b
0.2±0.00b
0.4
0.2
Mean values ± standard deviations. Superscripts in the same column with different
letters indicate significant differences (p < 0.05).
Low MIC value of 0.2 mg/mL was observed in pH 5.0, as expected for a
polyelectrolyte with positive charged amine radicals, improving the bind of macromolecule to
the negative charged plasmatic membrane of bacteria and/or interfering the replication nuclear
process and/or gene transcription, this explanation need more investigation (KRISHNAN,
2001; LIU, H. et al., 2004).
7.3.3 Integrity of target-cell membrane
It was observed an increased in the OD260 measures, suggesting the release of nucleic
acid material from E. coli cells in when chitosans were added at different concentrations, 0.5
and 1.0 mg/mL. The concentration of release of nucleic acids was affected by both chitosan
concentration and incubation time and the results were compared with the cellular growth)
(Fig. 1).
128
Figure. 1. The ratio release of nucleic acids and growth inhibition of E. coli cells. (A) Release of nucleic
acids evaluated by increments in the absorbance at 260nm and (B) growth of inhibition (%) of E. coli cells
harvested in LB medium at pH7.0. The white and black bars represent the growth inhibition at 0.5 and 1.0
mg/mL of chitosan solutions, respectively, in all panels. The numbers 1, 2 and 3 represent the CS-LMW, CSMMW and CS-MMW30 in all panels, respectively. The samples with same letter showed significance
difference, p<0.05.
129
The CS-LMW at the concentration of 0.5 mg/mL provoked a constant release of nucleic
acids and the inhibition of cell growth near 70% after 4h of chitosan exposition (figure 1,
panels A1 and B1). Chitosan treatment may cause minor damages to the cells keeping a
constant inhibition. The release of nucleic acids and inhibition of growth by CS-MMW and
CS-MMW30 seems to show similar and constant behavior (figure 1, panels A2, B2 and A3,
B3). It seems that these samples form aggregates that interfere in the measurement of nucleic
acids during the assay, because the encapsulation characteristic of chitosan aggregates nucleic
acids molecules. The AGUDELO; KREPLAK; TAJMIR-RIAHI (2016) reported that linkage
among DNA and chitosan occurs through electrostatic interactions between the polymer
cationic charged NH2 and negatively charged backbone phosphate groups. The phenomenon
of aggregation can be deduced from the PdI and ζ-potential measures (Table 1) where it was
observed a high % of inhibition but the concentration of nucleic acids is constant after 4 h of
incubation.
In general, the highest increase in OD260nm values provoked by CS-MMW and CSMMW30 at both concentrations was obtained after 1h incubation (figure 1, panels A2 and
A3), which is consistent with the results that was found under certain incubation times after
treating the R22579 strains of Xanthomonas with chitosan (WANG, Y. et al., 2012). At the
same time a growth inhibition was seen. After 4h incubation the % of inhibition is higher the
55% at all samples.
The difference in growth inhibition compared with the release of nucleic acids can be
discussed considering the molecular weight. The smallest macromolecule can be enter
through the plasmatic membrane altering the DNA transcription and killing more cells than
the largermolecules that do not reduce the transit of nutrients from medium and kills the cells
slowly but a relative growth of cells was detected.
Interestingly, the antibacterial mechanism of chitosan has been mainly attributed to the
interaction between positive charge chitosan and the negative charge bacterial membrane,
which may cause the leaking of low molecular weight compounds, nucleic acid and proteins
(DINA RAAFAT et al., 2008; LI, X.-F. et al., 2010). Indeed, various studies performed
recently have shown that this charge interaction can alter bacterial surface morphology and
increase membrane permeability, causing leakage of intracellular substances. That
phenomenon can reinforce the importance of the interaction between chitosan and bacterial
130
membrane (EATON et al., 2008; TANG, H. et al., 2010). The mode of action of chitosan
against E. coli cells, may, at least partially, be attributed to this charge interaction between
chitosan and bacteria. This indicated the complexity of the interactions between chitosan and
bacterial membranes.
7.4 PARTIAL CONCLUSIONS
In this study, the physical and chemical characteristics of chitosan nanoparticles
produced by ultrasound irradiation were evaluated and compared with its inhibition activity
towards E. coli in vitro. Experimental figures and data presented have provided several
evidences that chitosans NPs with low and medium molecular weights demonstrated strong
inhibition activity against E. coli, but the inhibition activity differed in pH and MW, within
the tested chitosans. The molecular weight and particle size/zeta potential allow an easy
manipulation of physicochemical properties of nanoparticles suitable for its application. More
experiments should help us go further to improve the understanding of the exact mode of
action of chitosan nanoparticles and its antibacterial activity.
Acknowledgments
The authors acknowledge the financial support from Fundação Carlos Chagas Filho de
Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ, Rio de Janeiro, Brazil), Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brasília, Brazil),
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brasília, Brazil) and
L.P.G. acknowledges CAPES (Programa Institucional de Bolsas de Doutorado Sanduíche no
Exterior, PDSE; Proc nº 18935/12-5).
131
8
DISCUSSÃO
Nos últimos anos, o conceito de desenvolvimento sustentável tem obtido atenção
política e social importante, devido à aplicação das "tecnologias verdes" e o uso de "produtos
verdes", que contribuem para a sustentabilidade através da diminuição da degradação
ambiental, estabelecendo melhores maneiras mais adequadas de sintetizar produtos
necessários para o desenvolvimento em diferentes áreas, ao mesmo tempo reduzindo os
impactos sobre o ambiente (DAHL et al., 2007).
A química verde explora técnicas de química e metodologias que reduzam ou
eliminem o uso ou geração de matérias-primas, produtos, subprodutos, solventes, reagentes,
etc., que são danosos à saúde humana ou ao ambiente (RAVEENDRAN et al., 2003).
Com isso vem crescendo a busca por alternativas mais ecológicas de produtos que se
enquadrem no conceito da “tecnologia verde”, a fim de substituir produtos derivados de
petróleo. As crescentes exigências da sociedade em matéria de sustentabilidade e proteção
ambiental deverão desencadear uma profunda transformação na produção industrial. Neste
sentido, o aproveitamento dos abundantes recursos de biomassa deverá preparar a sociedade
para uma bioeconomia.
Biopolímeros naturais são recursos adequados para biofabricação de materiais
ambientalmente
adequados
devido
às
características
de
biodegradabilidade,
biocompatibilidade, e abundância. A quitosana é um polissacarídeo obtido por Ndesacetilação da quitina, o seu precursor natural, que é extraído do exoesqueleto de crustáceos
e insetos (DEL AGUILA et al., 2012) é considerada uma alternativa para substituição de
derivados do petróleo na produção de embalagens, principalmente as destinadas à indústria de
alimentos e cosméticos, devido à capacidade de serem processados como hidrogéis,
membranas, micropartículas, nanopartículas coloidais (SHUKLA, SUDHEESH K. et al.,
2013).
Na ultima década, a quitosana tem sido explorada como material para produção de
nanopartículas, a fim de melhorar suas propriedades, devido as características diferenciadas
que as moléculas nessa escala podem apresentar, tais como seu caráter único como o seu
tamanho quântico pode resultar em nanopartículas de quitosana com atividades superiores,
apresentando melhor potencial mucoadesivo de natureza hidrofílica e, por causa disso; podem
132
ser utilizadas para a encapsulação de drogas, controlando o tempo de liberação e conferindo
maior estabilidade as drogas quando presentes no organismo.
Diante disso, novas formas de produção de nanopartículas vêm sendo desenvolvidas,
sempre baseados no conceito da “tecnologia verde”. A irradiação por ultrassom oferece um
potencial importante para a conversão de biomassa polimérica, tais como carboidratos, em
moléculas de menor massa molar (GOMES, LAIDSON P. et al., 2010; KANG et al., 2014;
SAVITRI et al., 2014). O uso de ultrassom tem demonstrado ser uma ferramenta simples e
econômica para a degradação de moléculas poliméricas. Através do controle da temperatura,
frequência, intensidade, tempo de sonicação e da concentração polimérica em solução é
possível controlar a extensão da degradação polimérica (KASAAI, MOHAMMAD R., 2008).
Nossos estudos exploraram o processo de sonicação a fim de produzir nanopartículas
de quitosana de diferentes tamanhos usando as mesmas condições de sonicação, mas variando
o tempo de irradiação de 5, 15 e 30 min.
As análises por DLS mostraram que a quitosana comercial,CHIT90 (NS), não tratada
possui três tipos predominantes de moléculas com Rh = 0.02, 0.18 e 19.5 µm, com
intensidades de 5.7, 46 e 48%, respectivamente. As imagens obtidas por AFM mostraram
quitosanas com baixo peso molecular (LMCH-NPs), na forma de esferas de nanopartículas
com diâmetros de 300 nm, sobre matriz com dimensões na escala micrométrica (HMCHmicrofibras). Foi possível observar pela técnica de SEM, a coexistência das LMCH-NPs e
HMCH-microfibras, nanofibras em torno de 150 nm e pequenas partículas esféricas variando
entre 10-20 nm (não observadas por AFM) (SOUZA, H. K. S. et al., 2013). A distribuição de
tamanhos de partículas observadas supõe a coexistência entre LMCH-NPs (Rh= 0.18 µm),
HMCH-microfibras (Rh= 19.5 µm) e NPs (Rh=0.02 µm).
A partir da exposição gradual da CHIT90 (NS) à sonicação, foi observada degradação
gradual da molécula de quitosana com o aumento do período de exposição à sonicação,
verificando se uma diminuição da intensidade de HMCH-microfibras acompanhada do
aumento concomitante na intensidade LMCH-NPs. As amostras NPs não apresentaram
variação relevante durante os intervalos de sonicação e a contribuição das microfibras no
espalhamento de luz diminuiu linearmente com o aumento do tempo de sonicação (ts).
Concomitantemente, o aumento linear da % da intensidade das LMCH-NPs também pode ser
notado confirmando que a degradação de HMCH-microfibra é responsável pelo aumento da
população de LMCH-NPs.
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A partir das soluções sonicadas (S) por ts= 5, 15 e 30 min foram feitas misturas entre
elas e a amostra original CHIT90 (NS) em diferentes proporções NS/S(ts) 7:3, 1:1 e 3:7. As
análises de AFM foram utilizadas para investigar as caraterísticas morfológicas das misturas
NS:S(ts). A diferença na topografia das diferentes amostras está correlacionada com as
diferentes misturas feitas e aos períodos de sonicação diferentes.
Em relação às misturas de S(5), a imagem da topografia da mistura 7:3 não se parece
com uma imagem típica da base de mica. A presença de uma densa película de quitosana é
sugerida, com uma espessura estimada em 2 nm a partir dos perfis de altura extraídos e acima
desta camada pode ser distinguida estruturas semelhantes à fibras não ramificadas com o
comprimento variando entre 0.5 e 1 μm, altura em torno de 4 nm e largura em torno de 40 nm.
Aparentemente estas parecem formar feixes mais grossos de cerca de 100 nm que se
organizam como redes. A alta densidade da película formada na camada inferior e a presença
de fibras sobre essa camada são compatíveis com as análises de AFM e SEM reportadas para
filmes constituídos apenas por NS CHIT90 e outros polímeros (SOUZA, H. K. S. et al., 2013)
quando preparados a partir de soluções diluídas (<30 μg·mL-1).
Dois outros tipos de estruturas podem ser identificados na camada mais externa,
partículas com diâmetro e altura em torno de 50 e 60 nm respectivamente e corpos de formas
não definidas com tamanho maior que 150 nm e altura em tono de 10 nm na mistura 7:3
NS/S(5). A partir de dados obtidos da análise de DLS foi observado um aumento de 1,8 vezes
no valor da % de intensidade dos picos referentes às partículas em torno de 200 nm LMCHNPs, conforme o período exposição da CHIT90 a sonicação é aumentado. Comparando a
altura individual das fibras e da película na camada inferior, supõe se que a última camada
pode ser formada por uma monocamada de um amontanhado de HMCH-microfibras ou um
conjugado de microfibras-NPs, sendo que a mistura avaliada (7:3 NS/S(5)) era constituída de
70% do material fibroso (NS CHIT90).
Pode ser visto nas imagens que as mudanças foram desencadeadas pelo aumento da
quantidade de S (5) nas misturas. Duas tendências são bem evidentes nas imagens: (i) embora
ocorra redução brusca das fibras, o aumento da presença de NPs esféricas na camada mais
externa pode ser notado e (ii) a densidade da película inferior sofre uma significante redução.
As análises de amplitude para as amostras S(15 e 30 min) não apresentam essas transições de
fibras para LMCH-NPs de maneira evidente.
Essas transições ficam mais claras nas análises dos dados de RMS e HAV. As amostras
NS/S(5) apresentam pequenas mudanças com adição de S(5) na mistura 3:7 devido à presença
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predominante das NPs na superfície, porém, as outras misturas 7:3 e 1:1 apresentaram uma
distribuição mais homogênea entre NPs e fibras, o que explica as mudanças discretas nessas
amostras.
Paras as amostras S(15 e 30 min) em contraste aos resultados obtidos por DLS, a
altura das partículas observadas nas imagens topográficas (de AFM) apresenta um aumento ao
ser acrescentado uma quantidade de S nas misturas. Tal aumento pode ser interpretado como
ação do fenômeno de agregação na formação dos filmes, sendo que esse processo pode
ocorrer em solução, antes da formação das películas ou através de um efeito dirigido a
secagem, que envolve a formação de microgotículas (KRISHNAN, 2001; SOUZA, H. K. S.
et al., 2013).
Em resumo foi observada uma transição morfológica a partir de um material rico em
fibras microscópicas para outros com aspecto cada vez mais nanoparticulado, que é
ocasionado pelo período de tempo (ts) crescente ou através da variação na proporção das
misturas NS:S(tS). A grande variedade de morfologias identificadas neste experimento mostra
que este sistema é promissor para a fabricação de biofilmes de quitosana com propriedades
ajustáveis.
A partir dessas misturas NS/S(ts) foram efetuados testes mecânicos durante o
cisalhamento dinâmico 0 - 100 rad s-1 onde somente no caso da amostra NS os módulos de
armazenamento (G’) e de perda (G’’) demonstram uma tendência a convergirem em valores
acima do limite analisado, caracterizando um comportamento viscoelástico (HANAOR et al.,
2012). Os dados obtidos para as amostras S(ts), de forma isolada, ilustram bem como o
aumento do ts afeta a magnitude de ambos os módulos diminuindo em toda gama de
frequências juntamente com o aumento da relação logG’’ – log G’. Esses efeitos já foram
avaliados em trabalho prévio e foi atribuído a continua diminuição na viscoelasticidade das
soluções que ocorre em paralelo com o aumento do tempo de irradiação (SOUZA, H. K. S. et
al., 2013). Esse comportamento indica uma diminuição contínua do emaranhado conforme a
degradação progride. A redução do emaranhado polimérico é atribuída principalmente à
diminuição da massa molar média, e aos efeitos correspondentes ao tamanho e morfologia dos
coloides (o que já foi investigado por DLS).
Essas conclusões são mais evidentes após análise das curvas de tan  vs. ω, que foram
calculados a partir da relação tan  = G´´/G´, onde os menores valores registrados foram
obtidos para CHIT90 (NS) o que confirma a maior elasticidade dessa amostra. A partir do
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aumento do ts de 0 a 30 min, a tan  aumenta progressivamente refletindo a redução da
elasticidade (aumentando o comportamento tipo liquido) das amostras sonicadas, sendo que
S(30) mostrou o maior valor. Além disso, foram feitos cálculos para as diferentes misturas, as
quais demonstraram que independente do ts aplicados, a redução da quantidade de CHIT90
(NS) nas misturas resultou na diminuição progressiva da elasticidade (aumentado o valor de
tan ) em toda gama de frequência e por consequência a diminuição da propriedade
viscoelástica. Assim, dependendo da ts aplicada e da proporção de misturas S (ts), podem ser
obtidas misturas exibindo diferentes propriedades viscoelástica, mas sempre dentro dos
limites definidos pela resposta dos seus componentes puros.
Assim propriedades viscoelásticas da quitosana comercial podem ser ajustadas através
da sua mistura com produtos da sua sonicação, verificando que a elasticidade das misturas
resultantes pode ser eficazmente reduzida pelo aumento da fração do componente sonicado
e/ou aumentando o tempo de exposição da CHIT90 sonicação.
A partir da combinação de dados de viscosidade aparente versus a taxa de
cisalhamento foi possível evidenciar um comportamento independente do tempo, típico de
fluidos não tixotrópicos, corroborando com o comportamento apresentado nos dados
dinâmicos, os maiores valores da ηA em toda gama da taxa de cisalhamento foi exibido pela
componente não sonicada, no caso das componentes sonicadas S (ts), a ηA mostrou uma
redução a partir dos altos níveis da amostra NS com o aumento do ts.
Foi observado que na região inicial das analises de cisalhamento efetuado paras as
amostras NS e S(ts), ocorre o rompimento dos entrelaçamentos intermoleculares por ação da
força de cisalhamento é bem equilibrada pela formação de novos entrelaçamentos, de modo a
não ocorrer mudança e a viscosidade no cisalhamento inicial é mantida. Entretanto o aumento
da 𝛾̇ , a perturbação do emaranhado torna se predominante sobre a formação e as moléculas
tendem a se alinhar na direção do fluxo o que resulta na diminuição da viscosidade. O
aumento do ts resulta em uma mudança dessa transição para valores mais elevados de 𝛾̇ e
também reduz progressivamente a contribuição da pseudoplasticidade. Isso está bem ilustrado
pela resposta quase que puramente Newtoniana registrada para a amostra S (30). Este
comportamento ocorre devido à diminuição significativa no tamanho dos coloídes de
quitosana (CHUNG et al., 2004; COTTER et al., 2005).
A pseudoplasticidade também foi diminuída nas misturas, conforme a diminuição da
contração da amostra CHIT90 (NS) nas mesmas. Em particular, enquanto as misturas de S (5)
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e (15) ainda exibiram algum tipo de decaimento da Lei das Potências, a mistura rica em S (30)
demonstrou uma pseudoplasticidade quase insignificante. De maneira geral, esses resultados
foram compatíveis com as conclusões descritas acima.
Fluidos poliméricos submetidos a transições de comportamento Newtoniano para
pseudoplástico podem ser modelados segundo as equações de Cross e Carreau (CHIELLINI;
SOLARO, 1996;
SOUSA et al., 2013;
TOMASIK; ZARANYIKA, 1995). Os dados
coletados durante a execução da taxa de cisalhamento estacionária foram ajustados aos
modelos simplificados acima. Em geral foi encontrado um bom ajuste para ambos os
modelos, que apresentaram baixos valores de RSS e MRD. Os valores ligeiramente menores
apresentados para o ajuste com o modelo de Cross, são um indicativo da melhor adequação
deste para descrever os sistemas estudados. Fluidos Newtonianos são tipicamente
caracterizados por valores de M e N que tendem a zero. Por tanto, a pseudoplasticidade
decrescente está associada ao aumento do ts, coincidindo perfeitamente com a diminuição
dessas constantes nas amostras sonicadas S (ts). No caso das misturas, as constantes
diminuem em paralelo ao aumento da fração nanoparticulada nas misturas.
Por outro lado, os parâmetros  e  podem ser considerados constantes de relaxação, o
que indica o aparecimento da pseudoplasticidade. A redução drástica destes parâmetros nas
componentes sonicadas (em relação aos valores máximos registrados para amostras NS)
confirma o efeito do tratamento ultrassônico sobre a resposta viscoelástica das soluções
coloidais estudadas, refletindo a redução progressiva da resposta pseudoplástica
caracterizando o aumento do comportamento tipo Newtoniano. No caso das misturas, os
valores registrados foram sempre menores do que os valores apresentados para a componente
pura sonicada e não sonicada.
Apesar de não existir uma ligação teórica entre os dados provenientes dos ensaios
mecânicos de cisalhamento estacionário e dinâmico, Cox e Merz descreveram uma boa
correlação empírica entre eles para certos sistemas. Mais especificamente apresentaram uma
boa correlação entre a viscosidade aparente (η) no cisalhamento estacionário e a viscosidade
complexa (|η*|) no cisalhamento oscilatório, em comparação a valores iguais de taxa de
cisalhamento e frequência, em soluções de polissacarídeos random-coil e de alguns polímeros
sintéticos (TANG, H. et al., 2010). No entanto, mesmo nesses casos, a regra não é aplicada a
altas frequências. A fim de estudar a aplicabilidade desta adaptação para investigar a relação
entre as viscosidades aparente (ηA), complexa (|η*|) e dinâmica (η’), essa regra foi aplicada
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nas amostras NS, S(ts) e nas misturas 1:1. Esta regra não pode ser aplicada na amostra 1:1 NS
S (5), onde um desvio entre as viscosidade pode ser observado em toda gama de cisalhamento
analisada, o qual pode ser ocasionado pela deterioração da estrutura devido: (i) ao efeito da
tensão de deformação aplicada ao sistema ou (ii) a diferentes tipos de rearranjos moleculares
que ocorrem através do cisalhamento aplicado que pode ser ocasionado por interações de
cadeia interpolímero específica, além da formação dos emaranhados (WRZYSZCZYNSKI et
al., 1995).
As diferenças entre as viscosidades sob o cisalhamento oscilatório e estacionário
foram claramente atenuadas quando ts foi aumentado. Por conseguinte, uma boa correlação
foi encontrada para S (15) e (30) e suas misturas correspondentes, dento do intervalo de 0,110 s-1. Estes resultados demonstram que o comportamento observado, ou seja,
soluções/dispersões de quitosana com caráter fortemente Newtoniano parecem seguir a regra
de Cox-Merz melhor do que aqueles que apresentam propriedades viscoelásticas maiores.
Embora neste trabalho caso essas diferenças pareçam ser principalmente determinadas pelas
mudanças da massa molar induzidas pelo tratamento com ultrassom.
Na ultima década, houve um grande interesse no desenvolvimento de utilização de
películas ativas de origem biológica, que são caracterizadas por possuírem atividade
antimicrobiana e antifúngica, a fim de melhorar a conservação de alimentos reduzindo a
utilização de conservantes químicos. Moléculas biologicamente ativas como a quitosana e
seus derivados tem um potencial significativo na indústria de alimentos. A fim de explorar
essa possibilidade, a partir das amostras NS, S(ts) e as misturas NS/S(ts) 7:3, 1:1 e 3:7, foram
produzidos filmes poliméricos homogêneos sem a adição de aditivos e plastificantes, através
do método knife coating.
A influência da composição dos filmes NS, S(ts) e NS/S(ts) sobre seu comportamento
mecânico foi estudada. Os filmes NS apresentaram valores de TS, E e Y de 82.8±2.7 MPa,
2.9±0.1% e 35.2±0.9 MPa respectivamente, como já relatado em trabalhos anteriores
(SOUZA, H. K. S. et al., 2013).
A incorporação de nanopartículas nas soluções de fibras leva a um resultado tal que os
filmes preparados com NS/S(ts) apresentaram uma resistência mecânica melhorada quando
comparada com os seus precursores S(ts). Diferentes comportamentos foram observados
dependendo da proporção da mistura usada. As amostras NS/S(15) apresentaram um ganho de
força na mistura com a proporção 7:3 e 1:1, a qual apresentou maior valor entre todas as
amostras. As amostras NS/S(5) apresentaram um ganho de força com o aumento da razão S(5)
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nas misturas, sendo que o a maior ganho foi na mistura com proporção 3:7, indicada pela
evolução do valor de TS. As formulações NS/S(30) não apresentaram variações expressivas.
As medidas de elasticidade (E) também sofreram variações dentre as formulações
testadas. As misturas NS/S(5 e 15) apresentaram valores de E maiores que seus precursores
NS e S(5 e 15), porém entre as misturas NS/S(15 e 30) não houve mudança significativa.
Comportamento diferente do observado nas misturas NS/S(5) que apresentaram valores
variados dentre as misturas.
O filme 7:3 e 1:1 NS/S(15) apresentaram melhor relação entre TS e E, apresentando
valores maiores que de CHIT90 e duas vezes maiores que seu percursor S(15). A partir desses
resultados pode se inferir que esse comportamento ocorre devido à característica da molécula
obtida após esse período de sonicação (15 min), que contém um número maior de cadeias
terminais livres que a amostra S(5), devido à fragmentação da cadeia pela sonicação. Esta
fragmentação permite maior possibilidade de interação com a matriz do filme (CHIT90), e
por ser um polímero com maior número de monômeros que as presentes na amostra S(30),
possuem um número maior de ligações do tipo β→1,4, o que confere maior resistência ao
filme (GOCHO et al., 2000).
Com relação ao Modulo de Young (Y) foram observadas propriedades distintas nos
três grupos: (i) os filmes preparados com a mistura NS/S(5) apresentou valores de Y mais
baixos do que os seus precursores NS e S; (ii) um comportamento oposto foi observado para
os filmes obtidos com NS/S(15) e (iii) os filmes NS/S(30) apresentaram valores de Y mais
baixos do que o filme NS e três vezes maior do que a amostra S(30). No entanto, para cada
grupo, o aumento na proporção de nanopartículas nas formulações dos filmes não causa
diferença em Y.
Estudos vêm demonstrando que nanopartículas de quitosana produzem novas
interações através dos grupamentos amina e as cadeias terminais presentes na molécula da
quitosana (ALBANNA et al., 2013;
OSTROWSKA-CZUBENKO; GIERSZEWSKA-
DRUŻYŃSKA, 2009). Em filmes produzidos a partir da mistura entre quitosana e alginato foi
demonstrada a presença de uma rede tridimensional formada por reticulação química por ação
das cadeias presentes na quitosana (OSTROWSKA-CZUBENKO; GIERSZEWSKADRUŻYŃSKA, 2009).
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Os parâmetros avaliados confirmam a hipótese de que as misturas entre fibras e
nanopartículas de quitosana resultam em filmes com melhores TS, E e Y. Neste sentido, nos
filmes de quitosana NS/S(ts) produzidos, as nanopartículas parecem ter um contato maior com
a matriz fibrosa dos filmes (CHIT90) devido a seu menor tamanho, favorecendo a
possibilidade de ligações químicas cruzadas entra as mesmas e por consequência alterando as
propriedades mecânicas dos filmes.
A possibilidade de controlar as características do filme que se deseja produzir através
da variação do tempo de sonicação e da proporção de mistura, urge como uma alternativa para
a substituição do uso de plastificantes, que podem ser transferidas da embalagem para o
produto embalado, representando uma ameaça a saúde, e ainda sendo uma alternativa para
substituição de produtos derivados de petróleo.
As propriedades de permeabilidade à água e absorção de água em filmes estão
associadas à difusão de moléculas através da matriz do filme, representando a relação de
equilíbrio entre o seu teor de umidade e da atividade da água (aw), que são necessários para
prever as propriedades de filmes em diferentes ambientes. A amostra NS apresentou maior
valor de WVP entre todas as amostras (14.2±1.1), seguido das amostras S(5), S(30) e S(15),
13.6±4.4, 7.8±2.0 e 7.9±0.7, respectivamente. Os valores apresentados para as misturas
NS/S(ts) apresentaram redução quando comparados com a amostra NS, concluindo que com a
adição das nanopartículas, independente do tempo de sonicação a WVP é melhorada.
Foi demonstrado recentemente que filmes preparados a partir de CHIT90, degradados
por sonicação, exibem menor WVP, devido à formação de pequenas partículas esféricas de
quitosana que devido aos seus domínios cristalinos aumentam a resistência à penetração do
solvente (SOUZA, H. K. S. et al., 2013). Estes dados corroboram com os resultados aqui
apresentados onde os filmes produzidos com amostras S(30), possuem a maior quantidade de
partículas menores dentre as amostras estudadas, e apresentam os menores valores de WVP.
Esse comportamento já foi observado em outros estudos, onde a adição de nanopartículas de
quitosana foi efetiva no intuito de melhorar o efeito de barreira em filmes de
poli(caprolactona) e metilcelulose (FABER et al., 2005; JANJARASSKUL; KROCHTA,
2010).
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Isotermas de sorção são procedimentos importantes para avaliar o efeito da
temperatura e da umidade relativa sobre propriedade dos filmes (LAI; PADUA, 1998). A
adição e remoção de água podem causar alterações na estrutura macromolecular do filme
(TORRES, 1994). O modelo de GAB foi usado para avaliação do comportamento das
isotermas das amostras de filme NS, S e NS/S(ts). Todas as misturas avaliadas apresentaram
um aumento de atividade de água 0,3-0,6 para os filmes produzidos com amostras puras. Os
resultados apresentados pelos filmes NS e S (ts) apresentaram se semelhantes aos resultados
já descritos por SOUZA, H. K. S. et al. (2013).
Como foi relatado nesse trabalho o processo de sonicação é capaz de produzir
nanopartículas de quitosana com tamanhos variados, os quais apresentam três sítios de
adsorção predominantes, os grupamentos hidroxila, amino e as cadeias terminais do polímero.
Com a diminuição do tamanho de partícula ocorre um aumento do número de cadeias
terminais por unidade de massa. Desse modo, novos sítios de adsorção são produzidos com a
degradação do polímero (GOCHO et al., 2000).
A quantidade de água envolvida na adsorção de uma monocamada é quantitativamente
(numa base seca) descrita pelos valores do parâmetro X0 e o parâmetro C representa a energia
entre a adsorção de uma ou mais monocamadas. As amostras NS/S(15 e 30) apresentaram
valores maiores de X0 que seus precursores, porem não apresentaram diferenças entre as
misturas indicando que estes filmes apresentam o maior número de sítios de adsorção do que
em uma monocamada de filmes secos.
Os resultados de sorção estão em linha com os resultados observados nas propriedades
mecânicas. Uma vez que as nanopartículas presentes em S(15) apresentam um tamanho maior
do que as presentes em S(30), e essa diferença de tamanho se reflete diretamente na
quantidade de ligações β→1,4, logo quanto menor a partícula menos ligações desse tipo ela
possui. Assim, a mistura de misturada NS com S(15) gera uma película mais resistente como
já mencionado. Diferente de quando adicionado S(30) às misturas, foram produzidos filmes
menos resistentes devido a essa deficiência de ligações β→1,4. Os filmes NS/S(30)
apresentaram valores pequenos para C e esse comportamento pode ser definido pelo fato de as
moléculas estarem menos fortemente ligadas aos sítios do polímero, como observado nas
análises de SEM. Os filmes apresentaram uma morfologia homogênea em que essas partículas
presentes em S(30) estão mais organizadas na matriz dos filmes, impedindo a ligação das
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moléculas de água. Esse comportamento está de acordo com os resultados de WVP em que
essas amostras apresentaram a maior redução desse parâmetro com adição das S(ts).
A determinação da cor é uma medida importante para a compreensão da imagem e
descrição do objeto, utilizada para conferir a avaliação da qualidade e inspeção de produtos
alimentares. Os filmes produzidos em geral, não apresentaram alterações de cor que
comprometessem a sua transparência.
Com o intuito de explorar o potencial antimicrobiano da quitosana e de nanopartículas
de quitosana, foi determinada a concentração mínima inibitória de amostras de quitosana
comercial (de baixo e médio pesos moleculares) e as nanopartículas oriundas da sonicação das
mesmas. O processo de sonicação foi eficaz na produção das nanopartículas como já foi
demonstrado anteriormente. As amostras não apresentaram alteração no potencial zeta e
tiveram uma polidispersividade variando entre 1.7 – 4.5 %. A atividade antimicrobiana foi
testada contra E. coli, onde foi observado que o valor de pH do meio de cultura interfere na
concentração mínima inibitória. A menor concentração inibitória observada foi conseguida
para a amostra de menor massa molar (CS-LMW30) com mínimo de 0.2mg/mL. No ensaio in
vitro foi possível observar que após 4 h de incubação com as diferentes amostras, todas
apresentaram inibição do crescimento acima de 55%.
Também pode ser observada, a
integridade da membrana plasmática das células, quando foi testada a amostra CS-LMW, que
apresentou um aumento na liberação de ácidos nucléicos com o decorrer do tempo
experimental. Esses resultados são um indicativo de que a quitosana pode estar rompendo a
membrana plasmática da célula e expondo o material genético da mesma, corroborando com
os resultados apresentados pela cinética de inibição.
142
9
CONLUSÕES
A ultrassonicação demonstrou ser capaz de degradar a quitosana através do controle
do tempo (ts) e da proporção de misturas (S(ts)/CHIT90) das soluções, resultando na
formação de soluções coloidais, sob condições controladas, sendo possível com isso ajustar as
propriedades viscoelásticas e a morfologia das soluções;
A partir das análises de AFM foi possível observar a presença de fibras e
nanopartículas nas amostras estudas, além de observar a morfologia dos filmes formados;
A mistura (NS/S) na proporção 1:1 preparada com quitosana sonicada por 15 min se
destaca como o sistema mais promissor devido à sua viscoelasticidade e morfologia
homogênea;
Os filmes biofilmes produzidos a partir das misturas coloidais são, flexíveis com
transparência e morfologia homogênea sem a adição de plastificantes.
As características mecânicas dos filmes e a permeabilidade ao vapor de água foram
ajustadas através do controle das proporções NS/S(ts), onde os filmes produzidos com as
proporções 7:3 e 1:1 NS/S(15) apresentaram um melhor equilíbrio entre as propriedades
mecânicas avaliadas.
Os filmes reforçados com as amostras S(30) apresentaram melhor propriedades de
permeabilidade ao vapor de água, sugerindo que essas moléculas menores se dispersão de
forma mais homogênea entre a matriz do filme formada por CHIT90, preenchendo os espaços
vazios e impedindo assim a passagem do vapor de água.
As preparações de misturas de quitosanas com distintas morfologias e massas
moleculares mostraram-se adequados como precursores alternativos para a fabricação de
filmes, com propriedades ajustáveis, para a embalagem de alimentos;
As concentrações mínimas inibitórias sofreram alterações com a mudança no pH do
meio de cultivo de E. coli.
Os valores do MBC são iguais ou maiores aos valores encontrados para MIC, para E.
coli, sendo a amostra CS-LMW30 apresentou maior atividade antimicrobiana, com MIC de
0.2 mg/mL em pH 5.0.
143
10
PESPECTIVAS FUTURAS
Determinar o potencial antimicrobiano dos filmes;
Determinar as mínimas concentrações inibitórias das nanopartículas (NPs) para
microrganismos Gram positivos (ex. Staphylococcus aureus);
Avaliar a expressão genica através de qPCR dos genes envolvidos na síntese da
membrana plasmática de patógenos alimentares (Escherichia coli e Staphylococcus aureus) ;
Determinar a massa molar das NPs por cromatografia de gel permeação associada aos
detectores de índice de refração, viscosidade capilar e espalhamento de luz de duplo laser (15°
e 90°);
Estudos dos mecânicos de inibição de crescimento de bactérias pela quitosana po
ensaios de agregação celular, acompanhados por microscopia ótica e de fluorescência;
Analisar a integridade da membrana celular dos microrganismos por microscopia
eletrônica de transmissão;
Associar as nanopartículas à moléculas com atividades biológicas, tais como quinonas
e peptídeos com atividade antimicrobiana;
Determinar a eficiência de carreamento e liberação das moléculas associadas às NPs,
(ex quinonas, peptídeos e ácidos nucleicos).
144
11
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164
ANEXO I
165
ANEXO II
Dept. de Química e Bioquímica da Universidade do Porto
Rua do Campo Alegre, 687, 4169-007, Porto, Portugal
23th September 2015
Dear Dr. Fiszman,
Attached to this letter you may find our response to the concerns raised by the reviewers in
the assessment of manuscript FOODHYD-D-15-00339. In the first place, we would like to
thank the reviewers for their excellent job and interesting suggestions which will certainly
contribute to improve the final quality of the manuscript. We agree with most of their claims
and the relevant changes have been introduced in the manuscript (please, see the attached list
of amendments).
In the attached rebuttal letter we reply to each specific claim, explaining the amendments
made to the original manuscript or why we did not found it necessary. For clarity, the list of
amendments is supplemented by a manuscript file with highlighted changes. A last revision
was also applied in terms of the English usage to make it further easier to the reader.
I do hope that, after the changes, the manuscript is now ready for publication in Food
Hydrocolloids
Sincerely yours,
Dr. José M. Campiña, Phone: (+351) 220402643,
Fax: (+351) 2260832959,
E-mail: [email protected]
166

universidade federal do rio de janeiro instituto de química programa

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